Phenoscope: an automated large‐scale phenotyping platform offering high spatial homogeneity

Increased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non‐destructive, real‐time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high‐throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro‐environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro‐environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large‐scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.     

Multidimensional optimization of promising antitumor xanthone derivatives

A promising antitumor xanthone derivative was optimized following a multidimensional approach that involved the synthesis of 17 analogues, the study of their lipophilicity and solubility, and the evaluation of their growth inhibitory activity on four human tumor cell lines. A new synthetic route for the hit xanthone derivative was also developed and applied for the synthesis of its analogues. Among the used cell lines, the HL-60 showed to be in general more sensitive to the compounds tested, with the most potent compound having a GI₅₀ of 5.1 μM, lower than the hit compound. Lipophilicity was evaluated by the partition coefficient (K_p) of a solute between buffer and two membrane models, namely liposomes and micelles. The compounds showed a log K_p between 3 and 5 and the two membrane models showed a good correlation (r² = 0.916) between each other. Studies concerning relationship between solubility and structure were developed for the hit compound and 5 of its analogues.     

Single‐molecule atomic force microscopy unravels the binding mechanism of a Burkholderia cenocepacia trimeric autotransporter adhesin

Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram‐negative bacteria. Prominent examples of TAAs are found in Burkholderia cepacia complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that Burkholderia cenocepacia TAAs mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single‐molecule atomic force microscopy to unravel the binding mechanism of BCAM0224, a prototype TAA from B. cenocepacia K56‐2. We show that the adhesin forms homophilic trans‐interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that BCAM0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that BCAM0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.     

Tripartite efflux pumps: energy is required for dissociation,but not assembly or opening of the outer membrane channel of the pump

The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE‐E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy‐independent. Cells expressing wild‐type MtrD, in an MtrCE background, were vancomycin‐insensitive, but moderately sensitive in an MtrCE‐E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE‐E434K vancomycin‐insensitive; imply that the pump–component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy‐dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE‐E434K is unable to do so, resulting in the vancomycin‐sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy‐independent, while both drug export and complex dissociation require active proton flux.     

High Concentration—Ethanol Fermentation of Raw Ground Corn

1-Pyrroline-5-carboxylate dehydrogenase was purified and crystallized from Bacillus sphaericus. The crystalline preparation gave a single band on polyacrylamide slab gel electrophoresis. The molecular weight of the enzyme was determined to be about 100,000 by gel filtration. The enzyme consists of two subunits which are identical in molecular weight (50,000), as judged on SDS slab gel electrophoresis. The enzyme shows an optimum pH of 6.5 to 7.0. Its activity was 8.1 times higher with NADP⁺ than with NAD ⁺, and the enzyme was stabilized by NADP⁺. The apparent Km values for l-l-pyrroline-5-carboxylate, NADP⁺ and NAD⁺ are 4.2 × 10^–5m (with NADP⁺), 9.5 × 10~⁶m and 2.5 × IO^-3 m, respectively. The enzyme reaction is irreversible. A simple method for the determination of l-ornithine involving ornithine ¿-aminotransferase and 1- pyrroline-5-carboxylate dehydrogenase from B. sphaericus was developed. A linear relationship was found between the absorbance at 340 nm and the amount of l-ornithine (50 ~ 400 nmol), and between the fluorescence and the amount of l-ornithine (0.2 ~ 10 nmol).     

Caudal Brainstem Raphe Nuclei: Neural Substrate for their Involvement in the Expression of Some Biological Rhythms

Nucleus raphe pallidus (RPa) lies ventrally in the caudal brainstem, where it is coextensive rostrally with the nucleus raphe magnus (RMg) and caudally with the nucleus raphe obscurus (ROb). Retrograde neuronal tracing studies of our laboratory, carried out in rats and presented elsewhere, with fluorogold, true-blue or fast-blue, iontophoretically injected or by crystalline deposit, along the RPa extent, displayed many labeled pericaria at the preoptic area (POA), as well as lateral (LH) and dorsomedial (DMH) hypothalamus; paraventricular nucleus (PVN) and dorsal (DR) and median (MnR) raphe nuclei among others structures. In addition, RPa, which projects to the intermediolateral column, has been demonstrated to bear relation to many of the somatic-visceral functions also reported for POA. Iontophoretic injections of PHA-L, an anterograde tracer, in the POA subnuclei, presented terminal and varicose labeled fibers in RPa, as well as in the RMg, ROb, paraventricular thalamic (PVA), PVN and supraoptic nucleus (SO), LH, subparaventricular zone (sPVZ) and locus coeruleus (LC). Interestingly, POA, PVA, PVN, LH and SO have been described as retino- and suprachiasmatic-recipients. Taken together, these neuronal connections between brainstem raphe nuclei and POA, the similarity of functions to which they are related, as well as connections with other retino-suprachiasmatic-recipient structures, suggest that these caudal brainstem raphe nuclei could be part of the output system for the expression of some biological rhythms.     

Hydrological attributes and rheophilic freshwater fish: stock assessment

The mathematical and statistical advances in fitting stock assessment models enabled the emergence of the paradigm of “integrated analysis”, which fits all available data to a single model of population dynamics that traditionally has total catch as the only forcing function of the system. This approach, however, allowed us to include, in a flexible way, the effect of hydrological regime as an additional forcing function. We tried to achieve this flexibility by making the annual recruitment rates and spawning biomass adjustable to the attributes of the hydrological cycle data. Our models showed that these attributes are influential in the population dynamics of Brycon hilarii of the Northern Pantanal, and their inclusion in the models allowed best partial fits (which considered fits only to the data components length- and age-compositions, CPUE of juveniles and adults) than the Base-case (without hydrological attributes). The best partial fits where obtained when the attributes “delay of floods” and “intensity of floods” were forcing the spawning biomass and the annual recruitment respectively, indicating that these characteristics of the population may be influenced by specific attributes of the water level. The use of integrated modeling contributed with the advancement of population ecology knowledge of rheophilic fish. It is recommended that freshwater fisheries management be integrated into the hydrology management.     

The emerging use of zebrafish to model metabolic disease

The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated in vivo. The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism’s utility as a model system for human metabolic diseases.     

Stage-Regulated GFP Expression in Trypanosoma cruzi: Applications from Host-Parasite Interactions to Drug Screening

Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.