Enantioselective Self-Replicators

Self-replicating molecules provide a simple approach for investigating fundamental processes in scenarios of the emergence of life. Although homochirality is an important aspect of life and of how it emerged, the effects of chirality on self-replicators have received only little attention so far. Here, we report several self-assembled self-replicators with enantioselectivity that emerge spontaneously and grow only from enantiopure material. These require a relatively small number of chiral units in the replicators (down to eight) and in the precursors (down to a single chiral unit), compared to the only other enantioselective replicator reported previously. One replicator was found to incorporate material of its own handedness with high fidelity when provided with a racemic mixture of precursors, thus sorting (L)- and (D)-precursors into (L)- and (D)-replicators. Systematic studies reveal that the presence or absence of enantioselectivity depends on structural features (ring size of the replicator) that appear to impose constraints on its supramolecular organization. This work reveals new aspects of the little researched interplay between chirality and self-replication and represents another step toward the de novo synthesis of life.</p

Lack of Cell Cycle Inhibitor p21 and Low CD4⁺ T Cell Suppression in Newborns After Exposure to IFN-β

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Characterization of the Molecular Interplay between <i>Moraxella catarrhalis</i> and Human Respiratory Tract Epithelial Cells

<div><p><i>Moraxella catarrhalis</i> is a mucosal pathogen that causes childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. During the course of infection, <i>M. catarrhalis</i> needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs, and consequently, efficient adhesion to epithelial cells is considered an important virulence trait of <i>M. catarrhalis</i>. By using Tn-seq, a genome-wide negative selection screenings technology, we identified 15 genes potentially required for adherence of <i>M. catarrhalis</i> BBH18 to pharyngeal epithelial Detroit 562 and lung epithelial A549 cells. Validation with directed deletion mutants confirmed the importance of <i>aroA</i> (3-phosphoshikimate 1-carboxyvinyl-transferase), <i>ecnAB</i> (entericidin EcnAB), <i>lgt1</i> (glucosyltransferase), and MCR_1483 (outer membrane lipoprotein) for cellular adherence, with ΔMCR_1483 being most severely attenuated in adherence to both cell lines. Expression profiling of <i>M. catarrhalis</i> BBH18 during adherence to Detroit 562 cells showed increased expression of 34 genes in cell-attached versus planktonic bacteria, among which ABC transporters for molybdate and sulfate, while reduced expression of 16 genes was observed. Notably, neither the newly identified genes affecting adhesion nor known adhesion genes were differentially expressed during adhesion, but appeared to be constitutively expressed at a high level. Profiling of the transcriptional response of Detroit 562 cells upon adherence of <i>M. catarrhalis</i> BBH18 showed induction of a panel of pro-inflammatory genes as well as genes involved in the prevention of damage of the epithelial barrier. In conclusion, this study provides new insight into the molecular interplay between <i>M. catarrhalis</i> and host epithelial cells during the process of adherence.</p> </div

Gene expression of <i>M. catarrhalis</i> BBH18 during adherence to Detroit 562 cells.

<p>A) Functional class distribution of genes differentially expressed in cell-attached relative to non-adherent (planktonic) <i>M. catarrhalis</i> (n = 4), depicted as number of genes per functional class category. B) Distribution of gene expression levels of known adhesins and structural components associated with adherence in cell-attached <i>M. catarrhalis</i>. Average log₂ microarrays signal intensities (SI) per gene were grouped into bins (1-log interval per bin), and the total number of genes within a bin is shown here.</p

Highly Penicillin-Resistant Multidrug-Resistant Pneumococcus-Like Strains Colonizing Children in Oeiras, Portugal: Genomic Characteristics and Implications for Surveillance▿ †

While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as “streptococci of the mitis group” that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006

Adhesion of directed gene deletion mutants to respiratory tract epithelial cells.

<p>Adherence of <i>aroA, ecnAB</i>, lgt1, and the putative lipoprotein MCR_1483 to both Detroit 562 (A) and A549 (B) cells was significantly attenuated compared to wild-type (WT). Adherence levels are expressed relative to WT (n ≥ 3) and shown as means and SEM. Statistical difference was determined with a Mann-Whitney test with * <i>P</i> < 0.05, ** <i>P</i> < 0.01, and *** <i>P</i> < 0.001.</p

Genes affecting adherence of <i>M. catarrhalis</i> to respiratory tract epithelial cells.

<p>Overlap between genes identified by Tn-seq as important for adherence of <i>M. catarrhalis</i> BBH18 to A549 and Detroit 562 epithelial cells.</p

An Evaluation of Using Population Pharmacokinetic Models to Estimate Pharmacodynamic Parameters for Propofol and Bispectral Index in Children

Background: To study propofol pharmacodynamics in a clinical setting a pharmacokinetic model must be used to predict drug plasma concentrations. Some investigators use a population pharmacokinetic model from existing literature and minimize the pharmacodynamic objective function. The purpose of the study was to determine whether this method selects the best-performing pharmacokinetic model in a set and provides accurate estimates of pharmacodynamic parameters in models for bispectral index in children after propofol administration. Methods: Twenty-eight children classified as American Society of Anesthesiologists physical status 1 who were given general anesthesia for dental treatment were studied. Anesthesia was given using target-controlled infusion of propofol based on the Kataria model. Propofol target plasma concentration was 7 mu g/ml for 15 min, followed by 1 mu g/ml for 15 min or until signs of awakening, followed by 5 mu g/ml for 15 min. Venous blood samples were taken 1, 2, 5, 10, and 15 min after each change in target. A classic pharmacokinetic-pharmacodynamic model was estimated, and the methodology of other studies was duplicated using pharmacokinetic models from the literature and (re-)estimating the pharmacodynamic models. Results: There is no clear relationship between pharmacokinetic precision and the pharmacodynamic objective function. Low pharmacodynamic objective function values are not associated with accurate estimation of the pharmacodynamic parameters when the pharmacokinetic model is taken from other sources. Conclusion: Minimization of the pharmacodynamic objective function does not select the most accurate pharmacokinetic model. Using population pharmacokinetic models from the literature instead of the 'true' pharmacokinetic model can lead to better predictions of bispectral index while incorrectly estimating the pharmacodynamic parameters

Adaptation of Bordetella pertussis to the respiratory tract

There is a lack of insight into the basic mechanisms by which Bordetella pertussis adapts to the local host environment during infection. We analysed B. pertussis gene expression in the upper and lower airways of mice and compared this to SO4-induced in vitro Bvg-regulated gene transcription. Approximately 30% of all genes were found to be differentially expressed between in vitro vs. in vivo conditions. This included several novel potential vaccine antigens that were exclusively expressed in vivo. Significant differences in expression profile and metabolic pathways were identified between the upper versus the lower airways, suggesting distinct antigenic profiles. We found high expression of several Bvg-repressed genes during infection and mouse vaccination experiments using purified protein fractions from both Bvg- and Bvg+ cultures demonstrated protection against intranasal B. pertussis challenge. This study provides novel insights into the in vivo adaptation of B. pertussis and may facilitate the improvement of pertussis vaccines

A novel quantitative PCR assay for the detection of Streptococcus pneumoniae using the competence regulator gene target comX

Streptococcus pneumoniae is responsible for an estimated 1.6 million deaths worldwide every year. While rapid detection and timely treatment with appropriate antibiotics is preferred, this is often difficult due to the amount of time that detection with blood cultures takes. In this study, a novel quantitative PCR assay for the detection of Streptococcus pneumoniae was developed. To identify novel targets, we analysed the pneumococcal genome for unique, repetitive DNA sequences. This approach identified comX, which is conserved and present in duplicate copies in Streptococcus pneumoniae but not in other bacterial species. Comparison with lytA, the current 'gold standard' for detection by quantitative PCR, demonstrated an analytic specificity of 100% for both assays on a panel of 10 pneumococcal and 18 non-pneumococcal isolates, but a reduction of 3.5 quantitation cycle values (± 0.23 sem), resulting in an increased analytical detection rate of comX. We validated our assay on DNA extracted from the serum of 30 bacteraemic patients who were blood culture positive for Streptococcus pneumoniae and 51 serum samples that were culture positive for other bacteria. This resulted in a similar clinical sensitivity between the comX and lytA assays (47%) and in a diagnostic specificity of 98.2 and 100% for the lytA and comX assays, respectively. In conclusion, we have developed a novel quantitative PCR assay with increased analytical sensitivity for the detection of Streptococcus pneumoniae, which may be used to develop a rapid bedside test for the direct detection of Streptococcus pneumoniae in clinical specimens