Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Making teeth to order: conserved genes reveal an ancient molecular pattern in paddlefish (Actinopterygii)

Ray-finned fishes (Actinopterygii) are the dominant vertebrate group today (+30 000 species, predominantly teleosts), with great morphological diversity, including their dentitions. How dental morphological variation evolved is best addressed by considering a range of taxa across actinopterygian phylogeny; here we examine the dentition of Polyodon spathula (American paddlefish), assigned to the basal group Acipenseriformes. Although teeth are present and functional in young individuals of Polyodon, they are completely absent in adults. Our current understanding of developmental genes operating in the dentition is primarily restricted to teleosts; we show that shh and bmp4, as highly conserved epithelial and mesenchymal genes for gnathostome tooth development, are similarly expressed at Polyodon tooth loci, thus extending this conserved developmental pattern within the Actinopterygii. These genes map spatio-temporal tooth initiation in Polyodon larvae and provide new data in both oral and pharyngeal tooth sites. Variation in cellular intensity of shh maps timing of tooth morphogenesis, revealing a second odontogenic wave as alternate sites within tooth rows, a dental pattern also present in more derived actinopterygians. Developmental timing for each tooth field in Polyodon follows a gradient, from rostral to caudal and ventral to dorsal, repeated during subsequent loss of teeth. The transitory Polyodon dentition is modified by cessation of tooth addition and loss. As such, Polyodon represents a basal actinopterygian model for the evolution of developmental novelty: initial conservation, followed by tooth loss, accommodating the adult trophic modification to filter-feeding.     

When the same is not the same: phenotypic variation reveals different plant ecological strategies within species occurring in distinct Neotropical savanna habitats

Variations in abiotic characteristics such as soil water availability and fertility impose different selective pressures on plant populations. This may produce intraspecific variability in functional traits, even at a fine spatial scale. We investigated whether functional traits related to water-use efficiency, resource-retention strategy, soil nutrient acquisition, and fire tolerance differ in species that occur in two different habitats of Brazilian Cerrado: rocky savannas and savanna woodlands. Rocky savannas occur over sandstone, quartzite outcrops and have shallow nutrient-poor and low-moisture rocky soils, while savanna woodlands occur over well-drained and deep soils with frequent fire regimes. We measured nine functional traits of 40 tree species that occur in both habitats. Rocky savanna individuals exhibited a greater water-use efficiency strategy. The resource-retention strategy in rocky savanna individuals was corroborated by lower adult maximum height. However, despite the lower nutrient availability in rocky savanna soils, we only detected lower leaf phosphorus content in individuals from this habitat. Furthermore, individuals from both habitats had equally thick bark, suggesting that the fire-defense strategy is related to a stable, rather than plastic trait. Overall, our results highlight the central role of contrasting soil water availability patterns in driving phenotypic plasticity within species. We conclude that savanna species are responding to water and nutrient availabilities, via plasticity in traits related to the resource-retention strategy, and preparing for future fires, via uniformly thick bark. Wide plant distribution in contrasting habitats is possible for species that can shift ecological strategies to survive in nutrient- and water-limited habitats such as rocky savannas.     

Part feeding at high-variant mixed-model assembly lines

The part feeding problem at automotive assembly plants deals with the timely supply of parts to the designated stations at the assembly line. According to the just-in-time principle, buffer storages at the line are frequently refilled with parts retrieved from a central storage area. In the industrial application at hand, this is accomplished by means of an internal shuttle system which supplies the various stations with the needed parts based on a given assembly sequence. The main objective is to minimize the required number of shuttle drivers. To solve this in-house transportation problem, a heuristic solution procedure is developed which is based on the decomposition of the entire planning problem into two stages. First, transportation orders are derived from the given assembly sequence. In the second stage, these orders are assigned to tours of the shuttle system taking transportation capacity restrictions, due dates and tour scheduling constraints into account. Numerical results show that the proposed heuristic solves even large-sized problem instances in short computational time. Benchmark comparisons with Kanban systems reveal the superiority of the proposed predictive part feeding approach.     

Testing multiple coordination constraints with a novel bimanual visuomotor task

The acquisition of a new bimanual skill depends on several motor coordination constraints. To date, coordination constraints have often been tested relatively independently of one another, particularly with respect to isofrequency and multifrequency rhythms. Here, we used a new paradigm to test the interaction of multiple coordination constraints. Coordination constraints that were tested included temporal complexity, directionality, muscle grouping, and hand dominance. Twenty-two healthy young adults performed a bimanual dial rotation task that required left and right hand coordination to track a moving target on a computer monitor. Two groups were compared, either with or without four days of practice with augmented visual feedback. Four directional patterns were tested such that both hands moved either rightward (clockwise), leftward (counterclockwise), inward or outward relative to each other. Seven frequency ratios (3∶1, 2∶1, 3∶2, 1∶1, 2∶3. 1∶2, 1∶3) between the left and right hand were introduced. As expected, isofrequency patterns (1∶1) were performed more successfully than multifrequency patterns (non 1∶1). In addition, performance was more accurate when participants were required to move faster with the dominant right hand (1∶3, 1∶2 and 2∶3) than with the non-dominant left hand (3∶1, 2∶1, 3∶2). Interestingly, performance deteriorated as the relative angular velocity between the two hands increased, regardless of whether the required frequency ratio was an integer or non-integer. This contrasted with previous finger tapping research where the integer ratios generally led to less error than the non-integer ratios. We suggest that this is due to the different movement topologies that are required of each paradigm. Overall, we found that this visuomotor task was useful for testing the interaction of multiple coordination constraints as well as the release from these constraints with practice in the presence of augmented visual feedback.     

The Population Decline and Extinction of Darwin’s Frogs

Darwin’s frogs (Rhinoderma darwinii and R. rufum) are two species of mouth-brooding frogs from Chile and Argentina. Here, we present evidence on the extent of declines, current distribution and conservation status of Rhinoderma spp.; including information on abundance, habitat and threats to extant Darwin’s frog populations. All known archived Rhinoderma specimens were examined in museums in North America, Europe and South America. Extensive surveys were carried out throughout the historical ranges of R. rufum and R. darwinii from 2008 to 2012. Literature review and location data of 2,244 archived specimens were used to develop historical distribution maps for Rhinoderma spp. Based on records of sightings, optimal linear estimation was used to estimate whether R. rufum can be considered extinct. No extant R. rufum was found and our modelling inferred that this species became extinct in 1982 (95% CI, 1980–2000). Rhinoderma darwinii was found in 36 sites. All populations were within native forest and abundance was highest in Chiloé Island, when compared with Coast, Andes and South populations. Estimated population size and density (five populations) averaged 33.2 frogs/population (range, 10.2–56.3) and 14.9 frogs/100 m² (range, 5.3–74.1), respectively. Our results provide further evidence that R. rufum is extinct and indicate that R. darwinii has declined to a much greater degree than previously recognised. Although this species can still be found across a large part of its historical range, remaining populations are small and severely fragmented. Conservation efforts for R. darwinii should be stepped up and the species re-classified as Endangered.     

Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes

We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-Tyr-Lys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRP1; Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-8376]. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRP1.