Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies' (ONT) cloud-based What's In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT's framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC's 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT's MinION portability, ease-of-use, and identification capability in remote locations.

Replacing amino acids in translation: expanding chemical diversity with non-natural variants.

Here, we describe a strategy for synthesis of peptides with multiple unnatural amino acids (UAAs) using in vitro translation. Our method involves removing a natural amino acid and replacing it with an UAA variant in a reconstituted translation system. Whereas other systems require engineered components or chemical synthesis to charge UAAs onto tRNAs prior to translation, our strategy utilizes the wild-type machinery and charging occurs concomitant with translation. The design of the system allows for easy quantification of the UAA's incorporation efficiency and fidelity.

Expression, purification, crystallization and preliminary X-ray studies of histamine dehydrogenase from Nocardioides simplex.

Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 A resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 A.

Breeding birds on small islands: island biogeography or optimal foraging?

1. We test MacArthur and Wilson's theory about the biogeography of communities on isolated habitat patches using bird breeding records from 16 small islands off the coasts of Britain and Ireland. 2. A traditional examination of patterns of species richness on these islands suggests that area and habitat diversity are important predictors, but that isolation and latitude have a negligible impact in this system. 3. Unlike traditional studies, we directly examine the fundamental processes of colonization and local extinction (cessation of breeding), rather than higher-order phenomena such as species richness. 4. We find that many of MacArthur and Wilson's predictions hold: colonization probability is lower on more isolated islands, and extinction probability is lower on larger islands and those with a greater diversity of habitats. 5. We also find an unexpected pattern: extinction probability is much lower on more isolated islands. This is the strongest relationship in these data, and isolation is the best single predictor of colonization and extinction. 6. Our results show that examination of species richness alone is misleading. Isolation has a strong effect on both of the dynamic processes that underlie richness, and in this system, the reductions in both colonization and extinction probability seen on more distant islands have opposing influences on species richness, and largely cancel each other out. 7. We suggest that an appropriate model for this system might be optimal foraging theory, which predicts that organisms will stay longer in a resource patch if the distance to a neighbouring patch is large. If nest sites and food are the resources in this system, then optimal foraging theory predicts the pattern we observe. 8. We advance the hypothesis that there is a class of spatial systems, defined by their scale and by the taxon under consideration, at which decision-making processes are a key driver of the spatiotemporal dynamics. The appropriate theory for such systems will be a hybrid of concepts from biogeography/metapopulation theory and behavioural ecology.