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.     

Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors

Additional evidence about the influence of the cell wall physical and chemical characteristics on protein extractability was determined by calculating the conversion factors of five different microalgae known to have different cell wall composition, and their protein extracts. The conversion factors obtained for crude rigid cell walled Chlorella vulgaris, Nannochloropsis oculata and Haematococcus pluvialis were 6.35, 6.28 and 6.25, respectively, but for their protein extracts the values were lower with 5.96, 5.86 and 5.63. On the other hand, conversion factor obtained for fragile cell walled microalgae Porphyridium cruentum and Athrospira platensis was 6.35 for the former and 6.27 for the latter, with no significant difference for their protein extract with 6.34 for the former and 6.21 for the latter. In addition, the highest hydro-soluble protein percentage recovered from total protein was for P. cruentum 80.3 % and A. platensis 69.5 % but lower for C. vulgaris with 43.3 %, N. oculata with 33.3 % and H. pluvialis with 27.5 %. The study spotted the light on the influence of the cell wall on evaluating the conversion factor and protein extractability. In addition, it showed the necessity of finding the conversion factor every time accurate protein quantification is required, and proved that there is not a universal conversion factor that can be recommended.     

Evaluation of the protein quality of Porphyridium cruentum

The amino acid profile of the red microalga Porphyridium cruentum and its protein extract have been determined in order to assess the nutritional quality of this biomass for human consumption. Total protein determined by elemental analysis represented 56 % of its dry weight. Hydro-soluble proteins extracted at pH 12 and 40 °C were analysed by the Lowry method giving 47 %, which represented 84 % of total protein per dry weight. The amino acid sequence of the biomass and the protein extract was composed of a set of essential (39 % for the former and 37 % for the latter) and non-essential amino acids (61 % for the former and 63 % for the latter) that compares favourably with the standard protein/amino acid requirements proposed by Food and Agricultural Organisation and World Health Organisation.     

Fluctuations in energy-related metabolites during the peri-parturition period in Lori-Bakhtiari ewes

This study describes the fluctuations in serum energy-related metabolites during a period of 2 weeks before, to 2 weeks after parturition in Lori-Bakhtiari ewes. The effect of parity was also studied. Blood profiles were determined in 60 healthy pregnant ewes with single (n = 30) and twin (n = 30) lambings. Blood was collected from each ewe on days 14 and 7 prepartum, and days 7 and 14 postpartum to determine the serum non-esterified fatty acid (NEFA), β-hydroxybutyrate (BHBA), glucose, cholesterol, blood urea nitrogen (BUN) and calcium (Ca) levels. The age of the ewes had no significant effect on the energy metabolism indicators. Serum NEFA, BHBA, glucose, BUN and calcium concentrations recorded peak levels 7 days before parturition. However, NEFA and BHBA recorded significant changes (P < 0.05) during the peri-parturition period. All metabolites changed significantly in ewes carrying twin-bearing ewes, compared to single-bearing ewes. Serum BHBA concentrations recorded positive correlations with the serum NEFA (P < 0.01) and cholesterol (P < 0.05), while blood glucose had negative correlations (P < 0.01) with NEFA, BHBA and Ca. Blood NEFA and BHBA recorded positive correlations (P < 0.05) with the BUN levels and negative correlations (P < 0.05) with Ca. The results showed that blood NEFA and BHBA levels are sensitive indicators of the energy balance during the peri-parturition period in ewes.     

Historical Shifts in Brazilian P. falciparum Population Structure and Drug Resistance Alleles

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.     

Acceleration Data Reveal Highly Individually Structured Energetic Landscapes in Free-Ranging Fishers (Pekania pennanti)

Investigating animal energy expenditure across space and time may provide more detailed insight into how animals interact with their environment. This insight should improve our understanding of how changes in the environment affect animal energy budgets and is particularly relevant for animals living near or within human altered environments where habitat change can occur rapidly. We modeled fisher (Pekania pennanti) energy expenditure within their home ranges and investigated the potential environmental and spatial drivers of the predicted spatial patterns. As a proxy for energy expenditure we used overall dynamic body acceleration (ODBA) that we quantified from tri-axial accelerometer data during the active phases of 12 individuals. We used a generalized additive model (GAM) to investigate the spatial distribution of ODBA by associating the acceleration data to the animals'' GPS-recorded locations. We related the spatial patterns of ODBA to the utilization distributions and habitat suitability estimates across individuals. The ODBA of fishers appears highly structured in space and was related to individual utilization distribution and habitat suitability estimates. However, we were not able to predict ODBA using the environmental data we selected. Our results suggest an unexpected complexity in the space use of animals that was only captured partially by re-location data-based concepts of home range and habitat suitability. We suggest future studies recognize the limits of ODBA that arise from the fact that acceleration is often collected at much finer spatio-temporal scales than the environmental data and that ODBA lacks a behavioral correspondence. Overcoming these limits would improve the interpretation of energy expenditure in relation to the environment.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.