Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield

The great need for more sustainable alternatives to fossil fuels has increased our research interests in algal biofuels. Microalgal cells, characterized by high photosynthetic efficiency and rapid cell division, are an excellent source of neutral lipids as potential fuel stocks. Various stress factors, especially nutrient‐starvation conditions, induce an increased formation of lipid bodies filled with triacylglycerol in these cells. Here we review our knowledge base on glycerolipid synthesis in the green algae with an emphasis on recent studies on carbon flux, redistribution of lipids under nutrient‐limiting conditions and its regulation. We discuss the contributions and limitations of classical and novel approaches used to elucidate the algal triacylglycerol biosynthetic pathway and its regulatory network in green algae. Also discussed are gaps in knowledge and suggestions for much needed research both on the biology of triacylglycerol accumulation and possible avenues to engineer improved algal strains.     

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: new insights into the understanding of the structure-biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(alpha1-2)Man(alpha1-O)Me, ConM/CGL:Man(alpha1-3)Man(alpha1-O)Me and ConM/CGL:Man(alpha1-4)Man(alpha1-O)Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R(factor) and R(free). Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.     

Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.     

Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle

The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca²⁺ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca²⁺ sparks, which are elementary SR Ca²⁺ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca²⁺ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere⁻¹ s⁻¹. The few Ca²⁺ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca²⁺ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca²⁺ flux underlying Ca²⁺ sparks detected in fully differentiated frog and fish fibers.     

Genetic analysis of two Portuguese populations of <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> by RAPD profiling

The clam Ruditapes decussatus is commercially important in the south of Portugal. The random amplified polymorphic DNA (RAPD) technique was applied to assess the genetic diversity and population structure of two Portuguese populations occurring in the Ria Formosa (Faro) and the Ria de Alvor, respectively. Twenty-five individuals of each population were investigated by RAPD profiles. Genetic diversity within populations, measured by the percentage of polymorphic loci (%P), varied between 68.57% (Alvor) and 73.88% (Faro). Shannon’s information index (H) and Nei’s gene diversity (h) were 0.281 and 0.176, respectively, for the Alvor population and 0.356 and 0.234 for the Faro population. Overall, genetic variation within R. decussatus populations was high. The total genetic diversity (H _T) was explained by a low variation between populations (G _ST = 0.145), which is consistent with high gene flow (N _m = 2.9). The analysis of molecular variance (AMOVA) showed that 65% of variability is within populations and 35% between populations (Φ_PT = 0.345; P ≥ 0.001). The value of Nei’s genetic distance was 0.0881, showing a low degree of population genetic distance, despite the different geographic origin. This is the first study on the population genetics of R. decussatus by RAPD technique. The results may be useful for restocking programs and aquaculture.     

Removal of pathogen and indicator microorganisms from liquid swine manure in multi-step biological and chemical treatment

Concern has greatly increased about the potential for contamination of water, food, and air by pathogens present in manure. We evaluated pathogen reduction in liquid swine manure in a multi-stage treatment system where first the solids and liquid are separated with polymer, followed by biological nitrogen (N) removal using nitrification and denitrification, and then phosphorus (P) extraction through lime precipitation. Each step of the treatment system was analyzed for Salmonella and microbial indicators of fecal contamination (total coliforms, fecal coliforms, and enterococci). Before treatment, mean concentrations of Salmonella, total coliforms, fecal coliforms, and enterococci were 3.89, 6.79, 6.23 and 5.73 log(10) colony forming units (cfu)/ml, respectively. The flushed manure contained 10,590 mg/l TSS, 8270 mg/l COD, 688 mg/l TKN and 480 mg/l TP, which were reduced >98% by the treatment system. Results showed a consistent trend in reduction of pathogens and microbial indicators as a result of each step in the treatment system. Solid-liquid separation decreased their concentrations by 0.5-1 log(10). Additional biological N removal treatment with alternating anoxic and oxic conditions achieved a higher reduction with average removals of 2.4 log(10) for Salmonella and 4.1-4.5 log(10) for indicator microbes. Subsequent P treatment decreased concentration of Salmonella and pathogen indicators to undetectable level (<0.3 log(10) cfu/ml) due to elevated process pH (10.3). Our results indicate that nitrification/denitrification treatment after solids separation is very effective in reducing pathogens in liquid swine manure and that the phosphorus removal step via alkaline calcium precipitation produces a sanitized effluent which may be important for biosecurity reasons.     

Evaluation of the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age: The DESiGN cluster randomised trial

BackgroundAntenatal detection and management of small for gestational age (SGA) is a strategy to reduce stillbirth. Large observational studies provide conflicting results on the effect of the Growth Assessment Protocol (GAP) in relation to detection of SGA and reduction of stillbirth; to the best of our knowledge, there are no reported randomised control trials. Our aim was to determine if GAP improves antenatal detection of SGA compared to standard care.Methods and findingsThis was a pragmatic, superiority, 2-arm, parallel group, open, cluster randomised control trial. Maternity units in England were eligible to participate in the study, except if they had already implemented GAP. All women who gave birth in participating clusters (maternity units) during the year prior to randomisation and during the trial (November 2016 to February 2019) were included. Multiple pregnancies, fetal abnormalities or births before 24⁺¹ weeks were excluded. Clusters were randomised to immediate implementation of GAP, an antenatal care package aimed at improving detection of SGA as a means to reduce the rate of stillbirth, or to standard care. Randomisation by random permutation was stratified by time of study inclusion and cluster size. Data were obtained from hospital electronic records for 12 months prerandomisation, the washout period (interval between randomisation and data collection of outcomes), and the outcome period (last 6 months of the study). The primary outcome was ultrasound detection of SGA (estimated fetal weight <10th centile using customised centiles (intervention) or Hadlock centiles (standard care)) confirmed at birth (birthweight <10th centile by both customised and population centiles). Secondary outcomes were maternal and neonatal outcomes, including induction of labour, gestational age at delivery, mode of birth, neonatal morbidity, and stillbirth/perinatal mortality. A 2-stage cluster–summary statistical approach calculated the absolute difference (intervention minus standard care arm) adjusted using the prerandomisation estimate, maternal age, ethnicity, parity, and randomisation strata. Intervention arm clusters that made no attempt to implement GAP were excluded in modified intention to treat (mITT) analysis; full ITT was also reported. Process evaluation assessed implementation fidelity, reach, dose, acceptability, and feasibility. Seven clusters were randomised to GAP and 6 to standard care. Following exclusions, there were 11,096 births exposed to the intervention (5 clusters) and 13,810 exposed to standard care (6 clusters) during the outcome period (mITT analysis). Age, height, and weight were broadly similar between arms, but there were fewer women: of white ethnicity (56.2% versus 62.7%), and in the least deprived quintile of the Index of Multiple Deprivation (7.5% versus 16.5%) in the intervention arm during the outcome period. Antenatal detection of SGA was 25.9% in the intervention and 27.7% in the standard care arm (adjusted difference 2.2%, 95% confidence interval (CI) −6.4% to 10.7%; p = 0.62). Findings were consistent in full ITT analysis. Fidelity and dose of GAP implementation were variable, while a high proportion (88.7%) of women were reached. Use of routinely collected data is both a strength (cost-efficient) and a limitation (occurrence of missing data); the modest number of clusters limits our ability to study small effect sizes.ConclusionsIn this study, we observed no effect of GAP on antenatal detection of SGA compared to standard care. Given variable implementation observed, future studies should incorporate standardised implementation outcomes such as those reported here to determine generalisability of our findings.Trial registrationThis trial is registered with the ISRCTN registry, ISRCTN67698474.

Matias C Vieira and colleagues evaluate the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age in the DESiGN cluster randomised trial.