Cooperative learning and elaborative interrogation: effects on children's learning

This experimental study examined the effects of cooperative learning and a question-answering strategy called elaborative interrogation ("Why is this fact true?") on the learning of factual information about familiar animals. Retention gains were compared across four study conditions: elaborative-interrogation-plus-cooperative learning, cooperative-learning, elaborative-interrogation, and reading-control. Sixth-grade students (n=68) were randomly assigned to the four conditions. All participants were given initial training and practice in cooperative learning procedures via three 45-minute sessions. After studying 36 facts about six animals, students' retention gains were measured via immediate free recall, immediate matched association, 30-day, and GO-day matched association tests. A priori comparisons were made to analyze the data. For immediate free recall and immediate matched association, significant differences were found between students in the three experimental conditions versus those in the control condition. Elaborative-interrogation and elaborativeinterrogation- plus-cooperative-learning also promoted longterm retention (measured via 30-day matched association) of the material relative to repetitive reading with elaborative-interrogation promoting the most durable gains (measured via GO-day matched association). The relationship between the types of elaborative responses and probability of subsequent retention was also examined. Even when students were unable to provide adequate answers to the why questions, learning was facilitated more so than repetitive reading. In general, generation of adequate elaborations was associated with greater probability of recall than was provision of inadequate answers. The findings of the study demonstrate that cooperative learning and the use of elaborative interrogation, both individually and collaboratively, are effective classroom procedures for facilitating children's learning of new information

Staphylococcal Small Colony Variants Have Novel Mechanisms for Antibiotic Resistance

Over the past 4 years, a variant subpopulation of Staphylococcus aureus has been characterized that is defective in electron transport. These organisms grow slowly and are typical of the previously described small colony variants (SCVs). Indeed, many earlier papers included data that are consistent with defective respiratory activity in SCVs. We present a hypothesis that serves as biochemical basis for the development of SCVs. These variants are particularly interesting because they have been associated with very persistent infections, and they are more resistant to many antibiotics than normal S. aureus. Because of their slow growth, atypical colonial morphology, and unusual biochemical profile, they are easily missed or misidentified in the clinical laboratory. This is of some significance, as this subpopulation is more resistant to antibiotics than the parent population from which they arose. When an infection is particularly resistant to therapy, persists for a long period, or fails to respond to apparently adequate antimicrobial therapy, clinicians and clinical laboratory personnel should consider special efforts to search for SCV

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient

AbstractThe lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (≥8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators

Bloodstream Infections Caused by Small-Colony Variants of Coagulase-Negative Staphylococci Following Pacemaker Implantation

Small-colony variants (SCVs) of Staphylococcus aureus cause persistent and relapsing infections. Relatively little is known regarding infections caused by SCVs of coagulase-negative staphylococci. We report two cases of pacemaker electrode infections due to SCVs of Staphylococcus epidermidis and Staphylococcus capitis. Sequence analysis of a portion of the 16S rRNA gene (16S rDNA) confirmed the identity of the staphylococcal species as S. capitis and S. epidermidis. Isolates from cultures of blood obtained over at least a 2-week interval were compared by pulsed-field gel electrophoresis and found to be clonal even though the colony morphology was very different. Analysis for auxotrophism revealed hemin dependencies for all isolated SCVs. The two cases have several clinical and laboratory characteristics (which are also seen with S. aureus SCV infections) and strongly suggest that SCVs of coagulase-negative staphylococci must be actively sought, because they grow very slowly and can be easily misse

Staphylococcus aureus small colony variants show common metabolic features in central metabolism irrespective of the underlying auxotrophism

In addition to the classical phenotype, Staphylococcus aureus may exhibit the small colony-variant (SCV) phenotype, which has been associated with chronic, persistent and/or relapsing infections. SCVs are characterized by common phenotypic features such as slow growth, altered susceptibility to antibiotic agents and pathogenic traits based on increased internalization and intracellular persistence. They show frequently auxotrophiesms mainly based on two different mechanisms: (i) deficiencies in electron transport as shown for menadione- and/or hemin-auxotrophs and (ii) thymidylate biosynthetic-defective SCVs. To get a comprehensive overview of the metabolic differences between both phenotypes, we compared sets of clinically derived menadione-, hemin- and thymidine-auxotrophic SCVs and stable site directed mutants exhibiting the SCV phenotype with their corresponding isogenic parental strains displaying the normal phenotype. Isotopologue profiling and transcriptional analysis of central genes involved in carbon metabolism, revealed large differences between both phenotypes. Labeling experiments with [U-13C6]glucose showed reduced 13C incorporation into aspartate and glutamate from all SCVs irrespective of the underlying auxotrophism. More specifically, these SCVs showed decreased fractions of 13C2-aspartate and glutamate; 13C3-glutamate was not detected at all in the SCVs. In comparison to the patterns in the corresponding experiment with the classical S. aureus phenotype, this indicated a reduced carbon flux via the citric acid cycle in all SCV phenotypes. Indeed, the aconitase-encoding gene (acnA) was found down-regulated in all SCV phenotypes under study. In conclusion, all SCV phenotypes including clinical isolates and site-directed mutants displaying the SCV phenotype were characterized by down-regulation of citric acid cycle activity. The common metabolic features in central carbon metabolism found in all SCVs may explain similar characteristics of the S. aure

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF

Retrospective analysis of antimicrobial resistance and bacterial spectrum of infection in Gabon, Central Africa

Background: Physicians depend on reliable information on the local epidemiology of infection and antibiotic resistance rates to guide empiric treatment in critically ill patients. As these data are scarce for Central Africa, we performed a retrospective analysis of microbiological findings from a secondary care hospital in Gabon. Methods: Microbiological reports from 2009 to 2012 were used to assess the non-susceptibility rates of the three most common isolates from six major types of infections (bloodstream, ear-eye-nose-throat, surgical site, skin and soft tissue, urinary tract and wound infection). Results: A high diversity of pathogens was found, but Staphylococcus aureus was predominant in the majority of infections. Overall, the three most prevalent pathogens in children were S. aureus (33.7%), Streptococcus pyogenes (8.1%) and Escherichia coli (4.5%) and in adults S. aureus (23.5%), E. coli (15.1%) and Klebsiella pneumoniae (7.4%). In total, 5.8% (n = 19) of all S. aureus isolates were methicillin resistant. The proportion of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae was 15.4% (n = 78), 49.4% of all K. pneumoniae were ESBL-producer (n = 42). Conclusion: The high diversity of potential pathogens and high resistance rates in Gram-negative bacteria challenge a rational empiric use of antibiotics. Countrywide continuous sentinel surveillance is therefore urgently needed.<br

Staphylococcus aureus pathogenicity in cystic fibrosis patients-results from an observational prospective multicenter study concerning virulence genes, phylogeny, and gene plasticity

Staphylococcus aureus and cystic fibrosis (CF) are closely interlinked. To date, however, the impact of S. aureus culture in CF airways on lung function and disease progression has only been elucidated to a limited degree. This analysis aims to identify bacterial factors associated to clinical deterioration. Data were collected during an observational prospective multi-center study following 195 patients from 17 centers. The average follow-up time was 80 weeks. S. aureus isolates (n = 3180) were scanned for the presence of 25 virulence genes and agr-types using single and multiplex PCR. The presence of specific virulence genes was not associated to clinical deterioration. For the agr-types 1 and 4, however, a link to the subjects' clinical status became evident. Furthermore, a significant longitudinal decrease in the virulence gene quantity was observed. Analyses of the plasticity of the virulence genes revealed significantly increased plasticity rates in the presence of environmental stress. The results suggest that the phylogenetic background defines S. aureus pathogenicity rather than specific virulence genes. The longitudinal loss of virulence genes most likely reflects the adaptation process directed towards a persistent and colonizing rather than infecting lifestyle

Genome-wide associations of gene expression variation in humans

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level