Field-based detection of bacteria using nanopore sequencing: Method evaluation for biothreat detection in complex samples.

From pathogen detection to genome or plasmid closure, the utility of the Oxford Nanopore Technologies (ONT) MinION for microbiological analysis has been well documented. The MinION's small footprint, portability, and real-time analytic capability situates it well to address challenges in the field of unbiased pathogen detection, as a component of a security investigation. To this end, a multicenter evaluation of the effect of alternative analytical approaches on the outcome of MinION-based sequencing, using a set of well-characterized samples, was explored in a field-based scenario. Three expert scientific response groups evaluated known bacterial DNA extracts as part of an international first responder (Chemical, Biological, Radiological) training exercise. Samples were prepared independently for analysis using the Rapid and/or Rapid PCR sequencing kits as per the best practices of each of the participating groups. Analyses of sequence data were in turn conducted using varied approaches including ONTs What's in my pot (WIMP) architecture and in-house computational pipelines. Microbial community composition and the ability of each approach to detect pathogens was compared. Each group demonstrated the ability to detect all species present in samples, although several organisms were detected at levels much lower than expected with some organisms even falling below 1% abundance. Several 'contaminant' near neighbor species were also detected, at low abundance. Regardless of the sequencing approach chosen, the observed composition of the bacterial communities diverged from the input composition in each of the analyses, although sequencing conducted using the rapid kit produced the least distortion when compared to PCR-based library preparation methods. One of the participating groups generated drastically lower sequencing output than the other groups, likely attributed to the limited computer hard drive capacity, and occasional disruption of the internet connection. These results provide further consideration for conducting unbiased pathogen identification within a field setting using MinION sequencing. However, the benefits of this approach in providing rapid results and unbiased detection must be considered along with the complexity of sample preparation and data analytics, when compared to more traditional methods. When utilized by trained scientific experts, with appropriate computational resources, the MinION sequencing device is a useful tool for field-based pathogen detection in mixed samples.

Impedance-based analysis of mu opioid receptor signaling and underlying mechanisms.

The mu opioid receptor is a G-protein coupled receptor able to signal through the Gαi /o class of G-protein and ß-arrestin pathways, stimulating down-stream effector pathways. Signaling bias occurs when different receptor agonists lead to different signaling outcomes. Traditionally these have been studied using end-point assays. Real-time cellular analysis platforms allow for the analysis of the holistic effects of receptor activation as an integrated output. While this allows for different ligands to be compared rapidly, the cellular mechanisms underlying the signal are not well described. Using an impedance based system, the impedance responses for two opioid ligands, morphine and DAMGO were examined. The impedance responses for these two agonists, while showing similar features, were distinct from each other. Some of the mechanisms underlying the mu opioid receptor coupled impedance changes were investigated. It was found that the response is a result of discrete cellular processes, including G-protein signaling and protein kinase phosphorylation.

Characterization of the regulatory and expression context of an alternative oxidase gene provides insights into cyanide-insensitive respiration during growth and development.

Alternative oxidase (AOX) is encoded in small multigene families in plants. Functional analysis of the Arabidopsis (Arabidopsis thaliana) alternative oxidase 1c (AtAOX1c) promoter, an AOX gene not induced by oxidative stress, indicated that regulation of expression was complex, with the upstream promoter region containing positive and negative response regions. Comparison to the promoter region of soybean (Glycine max) alternative oxidase 2b (GmAOX2b), another AOX gene not induced by oxidative stress, revealed that they contained seven sequence elements in common. All elements were active in the promoter region of AtAOX1c in suspension cells and in leaf tissue from Columbia and mutant plants, where a mitochondrial protein import receptor was inactivated. Analysis of coexpressed and putatively coregulated genes, the latter defined as containing five or more sequence elements functional in AtAOX1c, indicated that AtAOX1c was coregulated with components involved with cell division and growth. Consistent with this analysis, we demonstrated that site II elements, previously shown to regulate the proliferating cell nuclear antigen, are present in the upstream promoter region of AtAOX1c and were strong negative regulators of AtAOX1c expression. It was demonstrated that NDB4, a gene encoding an external NAD(P)H dehydrogenase, displayed strong coexpression with AtAOX1c. Overall, these results indicate that AtAOX1c is regulated by growth and developmental signals.

Analysis of the alternative oxidase promoters from soybean.

Alternative oxidase (Aox) is a nuclear-encoded mitochondrial protein. In soybean (Glycine max), the three members of the gene family have been shown to be differentially expressed during normal plant development and in response to stresses. To examine the function of the Aox promoters, genomic fragments were obtained for all three soybean genes: Aox1, Aox2a, and Aox2b. The regions of these fragments immediately upstream of the coding regions were used to drive beta-glucuronidase (GUS) expression during transient transformation of soybean suspension culture cells and stable transformation of Arabidopsis. The expression patterns of the GUS reporter genes in soybean cells were in agreement with the presence or absence of the various endogenous Aox proteins, determined by immunoblotting. Deletion of different portions of the upstream regions identified sequences responsible for both positive and negative regulation of Aox gene expression in soybean cells. Reporter gene analysis in Arabidopsis plants showed differential tissue expression patterns driven by the three upstream regions, similar to those reported for the endogenous proteins in soybean. The expression profiles of all five members of the Arabidopsis Aox gene family were examined also, to compare with GUS expression driven by the soybean upstream fragments. Even though the promoter activity of the upstream fragments from soybean Aox2a and Aox2b displayed the same tissue specificity in Arabidopsis as they do in soybean, the most prominently expressed endogenous genes in all tissues of Arabidopsis were of the Aox1 type. Thus although regulation of Aox expression generally appears to involve the same signals in different species, different orthologs of Aox may respond variously to these signals. A comparison of upstream sequences between soybean Aox genes and similarly expressed Arabidopsis Aox genes identified common motifs.