Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

ABSTRACT Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro . The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate

Malaria Risk in Travelers

Malaria risk around the world was assessed by using Swedish surveillance data from 1997 to 2003 with an extensive travel database as denominator

A prospective study of Helicobacter pylori in relation to the risk for pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>The relationship between <it>Helicobacter pylori </it>infection and pancreatic cancer has been investigated in three previous studies with contradictory results. The aim of the present study was to investigate the association between <it>H. pylori </it>seropositivity and the risk for pancreatic cancer in a nested case-control study within a population based cohort.</p> <p>Methods</p> <p>Selected birth-year cohorts (born 1921–1949) of residents in Malmö, Sweden, were invited to a health screening investigation. A total of 33 346 subjects participated. Cases with pancreatic cancer (n = 87) were matched to controls (n = 263) using age, sex and time for baseline investigation as matching variables. <it>H. pylori </it>serology was analysed in stored serum samples using an enzyme-linked immunosorbent assay. Odds ratios (OR) for pancreatic cancer were calculated with 95% confidence intervals (CI) using logistic regression.</p> <p>Results</p> <p><it>H. pylori </it>seropositivity was not associated with pancreatic cancer in the total cohort (adjusted OR 1.25 (0.75–2.09)). However, a statistically significant association was found in never smokers (OR 3.81 (1.06–13.63) adjusted for alcohol consumption) and a borderline statistically significant association was found in subjects with low alcohol consumption (OR 2.13 (0.97–4.69) adjusted for smoking).</p> <p>Conclusion</p> <p>We conclude that no association between <it>H. pylori </it>infection and the risk for pancreatic cancer was found in the total cohort. However, in never smokers and in subjects with low risk alcohol consumption, a positive <it>H. pylori </it>serology was associated with an increased risk for pancreatic cancer. These findings should be interpreted cautiously due to the limited number of cases in these subgroups.</p

Då fladdrade öronen : Pedagogers arbetssätt och uppfattningar om högläsning i grundsärskolan

Syftet med denna studie är att undersöka hur högläsning bedrivs som lärandeform på grundsärskolan. Genom intervjuer och observationer har fem lärares inställning och arbetssätt gällande högläsning studerats. Studien visar på att lärarna är medvetna om högläsningens positiva effekter. De är även måna om att elevernas åsikter och personliga erfarenheter ska få ett visst utrymme, det framkommer också att lärarna arbetar med högläsningen på ett sätt som kan stärka elevernas självförtroende. Samtliga lärare betonar vikten av inlevelse för att främja lärandet vid högläsning. Kopplat till den litteratur vi tagit del av kan lärarnas arbetssätt främja elevernas förmåga att associera, tänka fritt och se samband. Genom detta kan även deras språkliga förmågor utvecklas samtidigt som de får känna att deras åsikter är av värde. I vissa fall fann vi dock att högläsningen kunde tillvaratas på ett ännu mer gynnsamt sätt; lärarna drog inte nytta av lärandeformens fulla potential. Vi fann emellertid att lärarna i övervägande fall gjorde väl grundade didaktiska val angående högläsning.

Då fladdrade öronen : Pedagogers arbetssätt och uppfattningar om högläsning i grundsärskolan

Syftet med denna studie är att undersöka hur högläsning bedrivs som lärandeform på grundsärskolan. Genom intervjuer och observationer har fem lärares inställning och arbetssätt gällande högläsning studerats. Studien visar på att lärarna är medvetna om högläsningens positiva effekter. De är även måna om att elevernas åsikter och personliga erfarenheter ska få ett visst utrymme, det framkommer också att lärarna arbetar med högläsningen på ett sätt som kan stärka elevernas självförtroende. Samtliga lärare betonar vikten av inlevelse för att främja lärandet vid högläsning. Kopplat till den litteratur vi tagit del av kan lärarnas arbetssätt främja elevernas förmåga att associera, tänka fritt och se samband. Genom detta kan även deras språkliga förmågor utvecklas samtidigt som de får känna att deras åsikter är av värde. I vissa fall fann vi dock att högläsningen kunde tillvaratas på ett ännu mer gynnsamt sätt; lärarna drog inte nytta av lärandeformens fulla potential. Vi fann emellertid att lärarna i övervägande fall gjorde väl grundade didaktiska val angående högläsning.

Environmental microbial communities living under very high antibiotic selection pressure

This book explores the role that environmental distribution of antimicrobial resistance genes and antibiotics plays in ecosystem and human health. The text features a multi-disciplinary framework that connects microbiology, environmental toxicology, and chemistry to assess human and ecological risk associated with exposure to antibiotics or antibiotic resistance genes as environmental contaminants. It also considers alternate uses and functions for antimicrobial compounds other than those intended for medicinal purposes in humans, animals, and fish. Recognizing the connectivity between overlapping complex systems, the book discusses the subject from the perspective of an ecosystem approach

Exploring the microbial resistome in river sediments exposed to extraordinary high levels of antibiotics

The rapid development and propagation of antibiotic resistance in pathogenic and opportunistic bacteria is a major threat to public health worldwide. The phenomenon has been widely studied in the clinical setting, but comparatively little is known about the prevalence and diversity of antibiotic resistance in communities of environmental bacteria, often referred to as the environmental resistome. As the external environment may function as a reservoir of resistance genes to human pathogens, we are interested in how environmental bacteria are affected by antibiotic pollution. We have previously isolated microbial DNA from river sediments taken up- and downstream from a water treatment plant that processes waste water from several pharmaceutical plants producing antibiotics. In a previous study, we used deep sequencing to identify unprecedented frequencies of known resistance genes to several classes of antibiotics in these samples. In this study, we aim to functionally characterize the resistome in a more open and exploratory way by screening genomic DNA libraries transformed into sensitive hosts. To generate the libraries, several experimental strategies were explored, including mechanical shearing and enzymatic digestion of the isolated DNA followed by blunt- or sticky end cloning into different plasmids, subsequently transformed into sensitive E. coli. Pros and cons of the different strategies will be discussed along with preliminary results of the screening against selected antibiotics