Using unmanned aerial vehicle‐based multispectral,RGB and thermal imagery for phenotyping of forest genetic trials: A case study in Pinus halepensis

The assessment of genetic differentiation in functional traits is fundamental towards understanding the adaptive characteristics of forest species. While traditional phenotyping techniques are costly and time‐consuming, remote sensing data derived from cameras mounted on unmanned aerial vehicles (UAVs) provide potentially valid high‐throughput information for assessing morphophysiological differences among tree populations. In this work, we test for genetic variation in vegetation indices (VIs) and canopy temperature among populations of Pinus halepensis as proxies for canopy architecture, leaf area, photosynthetic pigments, photosynthetic efficiency and water use. The interpopulation associations between vegetation properties and above‐ground growth (stem volume) were also assessed. Three flights (July 2016, November 2016 and May 2017) were performed in a genetic trial consisting of 56 populations covering a large part of the species range. Multispectral (visible and near infrared wavelengths), RGB (red, green, blue) and thermal images were used to estimate canopy temperature and vegetation cover (VC) and derive several VIs. Differences among populations emerged consistently across flights for VC and VIs related to leaf area, indicating genetic divergence in crown architecture. Population differences in indices related to photosynthetic pigments emerged only in May 2017 and were probably related to a contrasting phenology of needle development. Conversely, the low population differentiation for the same indices in July 2016 and November 2016 suggested weak interpopulation variation in the photosynthetic machinery of mature needles of P. halepensis. Population differences in canopy temperature found in July 2016 were indicative of variation in stomatal regulation under drought stress. Stem volume correlated with indices related to leaf area (positively) and with canopy temperature (negatively), indicating a strong influence of canopy properties and stomatal conductance on above‐ground growth at the population level. Specifically, a combination of VIs and canopy temperature accounted for about 60% of population variability in stem volume of adult trees. This is the first study to propose UAV remote sensing as an effective tool for screening genetic variation in morphophysiological traits of adult forest trees.     

Escherichia coli “TatExpress” strains export several g/L human growth hormone to the periplasm by the Tat pathway

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.     

Split-enzyme fragment as a single affinity tag that enables protein expression,purification, and functional assays

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.     

Do phytogeographic patterns reveal biomes or biotic regions?

We present the largest comparative biogeographical analysis that has complete coverage of Australia's geography (20 phytogeographical subregions), using the most complete published molecular phylogenies to date of large Australian plant clades (Acacia, Banksia and the eucalypts). Two distinct sets of areas within the Australian flora were recovered, using distributional data from the Australasian Virtual Herbarium (AVH) and the Atlas of Living Australia (ALA): younger Temperate, Eremaean and Monsoonal biomes, and older southwest + west, southeast and northern historical biogeographical regions. The analyses showed that by partitioning the data into two sets, using either a Majority or a Frequency method to select taxon distributions, two equally valid results were found. The dataset that used a Frequency method discovered general area cladograms that resolved patterns of the Australian biomes, whereas if widespread taxa (Majority method, with >50% of occurrences outside a single subregion) were removed the analysis then recovered historical biogeographical regions. The study highlights the need for caution when processing taxon distributions prior to analysis as, in the case of the history of Australian phytogeography, the validity of both biomes and historical areas have been called into question.     

Climate alters the movement ecology of a non‐migratory bird

Global climate change is causing increased climate extremes threatening biodiversity and altering ecosystems. Climate is comprised of many variables including air temperature, barometric pressure, solar radiation, wind, relative humidity, and precipitation that interact with each other. As movement connects various aspects of an animal''s life, understanding how climate influences movement at a fine‐temporal scale will be critical to the long‐term conservation of species impacted by climate change. The sedentary nature of non‐migratory species could increase some species risk of extirpation caused by climate change. We used Northern Bobwhite (Colinus virginianus; hereafter bobwhite) as a model to better understand the relationship between climate and the movement ecology of a non‐migratory species at a fine‐temporal scale. We collected movement data on bobwhite from across western Oklahoma during 2019–2020 and paired these data with meteorological data. We analyzed movement in three different ways (probability of movement, hourly distance moved, and sinuosity) using two calculated movement metrics: hourly movement (displacement between two consecutive fixes an hour apart) and sinuosity (a form of tortuosity that determines the amount of curvature of a random search path). We used generalized linear‐mixed models to analyze probability of movement and hourly distance moved, and used linear‐mixed models to analyze sinuosity. The interaction between air temperature and solar radiation affected probability of movement and hourly distance moved. Bobwhite movement increased as air temperature increased beyond 10°C during low solar radiation. During medium and high solar radiation, bobwhite moved farther as air temperature increased until 25–30°C when hourly distance moved plateaued. Bobwhite sinuosity increased as solar radiation increased. Our results show that specific climate variables alter the fine‐scale movement of a non‐migratory species. Understanding the link between climate and movement is important to determining how climate change may impact a species’ space use and fitness now and in the future.     

SketchBio: a scientist’s 3D interface for molecular modeling and animation

Background

Because of the difficulties involved in learning and using 3D modeling and rendering software, many scientists hire programmers or animators to create models and animations. This both slows the discovery process and provides opportunities for miscommunication. Working with multiple collaborators, a tool was developed (based on a set of design goals) to enable them to directly construct models and animations.

Results

SketchBio is presented, a tool that incorporates state-of-the-art bimanual interaction and drop shadows to enable rapid construction of molecular structures and animations. It includes three novel features: crystal-by-example, pose-mode physics, and spring-based layout that accelerate operations common in the formation of molecular models. Design decisions and their consequences are presented, including cases where iterative design was required to produce effective approaches.

Conclusions

The design decisions, novel features, and inclusion of state-of-the-art techniques enabled SketchBio to meet all of its design goals. These features and decisions can be incorporated into existing and new tools to improve their effectiveness.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-334) contains supplementary material, which is available to authorized users.     

Characterization of initiation factor 3 from wheat germ. 2. Effects of polyclonal and monoclonal antibodies on activity

Rabbit polyclonal antibodies to wheat germ initiation factor 3 (eIF-3) were obtained and were shown to react strongly with 4 of the 10 subunits of eIF-3 (pp116, pp87, pp56, and pp36). Two mouse monoclonal antibodies were obtained, one of which reacts specifically with pp87 and one of which reacts specifically with pp36. Highly purified anti-pp87 has no effect on the activity of eIF-3. Highly purified polyclonal antibodies and anti-pp36 inhibit the ability of eIF-3 to support polypeptide synthesis in vitro and the ability of eIF-3 to support mRNA binding to 40S ribosomal subunits. These results provide additional evidence that pp116, pp87, and pp36 are in exposed positions in the eIF-3 particle and that pp36 is essential for activity.