Structure and evolution of chlorate reduction composite transposons.

UnlabelledThe genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algae ACDC) and three previously isolated strains (Ideonella dechloratans, Pseudomonas sp. strain PK, and Dechloromarinus chlorophilus NSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificans BC plasmid pALIDE01 and Pseudomonas chloritidismutans AW-1). De novo assembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualization. Phylogenetic analysis places chlorate reductase within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of enzymes with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding chlorite dismutase (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate that cld has been mobilized at least once from a perchlorate reducer to build chlorate respiration.ImportanceGenome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction

Transposon and deletion mutagenesis of genes involved in perchlorate reduction in Azospira suillum PS.

UnlabelledAlthough much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism.ImportanceAlthough perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island, which is potentially capable of moving between organisms. Some of the genes identified are known to be directly involved in the metabolism of perchlorate, but other new genes likely regulate the metabolism in response to environmental signals. This work has uncovered new questions about the regulation, energetics, and evolution of perchlorate reduction but also presents the tools to address them

Reducing Car Use Amongst Older Drivers

Our work comprised a pilot study exploring potential means to support older people to reduce their car use. This group is under-represented in behaviour change research in transport, which often focuses on delaying the take up of driving or other critical stages in the life course such as having children. Indeed, research on older drivers is largely dominated by work exploring the potential negative impacts on their physical and mental health of driving cessation. Nonetheless, given the demands of the climate emergency and the scale of the requirement to reduce car use implied in any credible decarbonisation pathway, all sections of society will have to change their travel behaviour, at least to some extent. It is our contention that research into how this can be achieved for older drivers is not only a necessary component of informing wider car use reduction behaviour change strategies, but also that older age groups have a crucial role to play in signalling the need for change to others

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

UnlabelledDespite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth.ImportanceThe ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism

Joint Angle Calculations using Motion Capture and Deep Learning Pose Estimation while Running

Marker based motion capture is currently the most accurate method of measuring human kinematics; however, it is expensive and is often limited to lab environments making it unsuitable for many applications. Two-dimensional methods are available through open source code, but it is unclear which of these methods provides the greatest accuracy. PURPOSE: The purpose of this study is to quantify the accuracy of pose estimation from a monocular electro-optical sensor with deep learning to infer segment end points and pose estimation utilizing two open-source code approaches. METHODS: One subject ran at 6.5 m/s for 15 s while being recorded with Vicon Nexus and an iPhone both running at 240 Hz. Visual 3D computed joint angles from the marker data. The iPhone view was placed perpendicular to the sagittal plane. Deep learning algorithms produced 2D pose information that was translated into hip, knee, and ankle sagittal plane joint angles. Pearson r correlations compared MediaPipe and OpenPose joint angle estimations through 15 s of running to the motion capture data. RESULTS: Markerless methods showed correlation values compared with Visual 3D of hip (MediaPipe = 0.968, OpenPose = 0.975), knee (MediaPipe = 0.983, OpenPose = 0.964), and ankle (MediaPipe = 0.928, OpenPose = 0.904). Both markerless methods showed limitations on predicting maximum flexion and extension angles. Although the correlation values were high, in practice these differences in maximum range of motion may impact any future interpretation of data. CONCLUSION: Care should be taken when utilizing extreme joint angles when using deep learning algorithms. Although at this point the open source methods are not as accurate as marker based motion capture they could enable the collection of data from a larger population of people given the ease of data collection, this could facilitate crowd sourced data collection with much larger sample sizes than are traditionally feasible

Comparison of single-layer and double-layer anti-reflection coatings using laser-induced damage threshold and photothermal common-path interferometry

The dielectric thin-film coating on high-power optical components is often the weakest region and will fail at elevated optical fluences. A comparison of single-layer coatings of ZrO2, LiF, Ta2O5, SiN, and SiO2 along with anti-reflection (AR) coatings optimized at 1064 nm comprised of ZrO2 and Ta2O5 was made, and the results of photothermal common-path interferometry (PCI) and a laser-induced damage threshold (LIDT) are presented here. The coatings were grown by radio frequency (RF) sputtering, pulsed direct-current (DC) sputtering, ion-assisted electron beam evaporation (IAD), and thermal evaporation. Test regimes for LIDT used pulse durations of 9.6 ns at 100 Hz for 1000-on-1 and 1-on-1 regimes at 1064 nm for single-layer and AR coatings, and 20 ns at 20 Hz for a 200-on-1 regime to compare the //ZrO2/SiO2 AR coating