Solving the Jigsaw puzzle of phytosterol diversity by a novel sterol methyltransferase from Zea mays.

Phytosterols are vital structural and regulatory components in plants. Zea mays produces a series of phytosterols that are specific to corn. However, the underline biosynthetic mechanism remains elusive. In this study, we identified a novel sterol methyltransferase from Z. mays (ZmSMT1-2) which showed a unique feature compared with documented plant SMTs. ZmSMT1-2 showed a substrate preference for cycloartenol. Using S-adenosyl-L-methionine (AdoMet) as a donor, ZmSMT1-2 converted cycloartenol into alkylated sterols with unique side-chain architectures, including Δ25(27) (i.e., cyclolaudenol and cycloneolitsol) and Δ24(25) (i.e., cyclobranol) sterols. Cycloneolitsol is identified as a product of SMTs for the first time. Our discovery provides a previously untapped mechanism for phytosterol biosynthesis and adds another layer of diversity of sterol biosynthesis.

Control Phytophagous Nematodes By Engineering Phytosterol Dealkylation Caenorhabditis elegans as a Model.

Plant-parasitic nematodes ingest and convert host phytosterols via dealkylation to cholesterol for both structural and hormonal requirements. The insect 24-dehydrocholesterol reductase (DHCR24) was shown in vitro as a committed enzyme in the dealkylation via chemical blocking. However, an increased brood size and ovulation rate, instead compromised development, were observed in the engineered nematode Caenorhabditis elegans where the DHCR24 gene was knocked down, indicating the relationship between DHCR24 and dealkylation and their function in nematodes remains illusive. In this study, a defect in C. elegans DHCR24 causes impaired growth of the nematode with sitosterol (a major component of phytosterols) as a sole sterol source. Plant sterols with rationally designed structure (null substrates for dealkylation) can't be converted to cholesterol in wild-type worms, and their development was completely halted. This study underpins the essential function of DHCR24 in nematodes and would be beneficial for the development of novel nematocidal strategies.

Diversity of Phytosterols in Leaves of Wild Brassicaceae Species as Compared to Brassica napus Cultivars: Potential Traits for Insect Resistance and Abiotic Stress Tolerance.

Phytosterols are natural compounds found in all higher plants that have a wide variety of roles in plant growth regulation and stress tolerance. The phytosterol composition can also influence the development and reproductive rate of strict herbivorous insects and other important agronomic traits such as temperature and drought tolerance in plants. In this study, we analysed the phytosterol composition in 18 Brassica napus (Rapeseed/canola) cultivars and 20 accessions belonging to 10 related wild Brassicaceae species to explore diverse and novel phytosterol profiles. Plants were grown in a controlled phytotron environment and their phytosterols were analysed using a saponification extraction method followed by GC-MS from the leaf samples. The B. napus cultivars showed slight diversity in eight phytosterols (>0.02%) due to the genotypic effect, whereas the wild accessions showed significant variability in their phytosterol profiles. Of interest, a number of wild accessions were found with high levels of campesterol (HIN20, HIN23, HUN27, HIN30, SARS2, and UPM6563), stigmasterol (UPM6813, UPM6563, ALBA17, and ALBA2), and isofucosterol (SARS12, SAR6, and DMU2). These changes in individual phytosterols, or ratios of phytosterols, can have a significant implication in plant tolerance to abiotic stress and plant insect resistance properties, which can be used in breeding for crop improvement.

Time-restricted eating with or without low-carbohydrate diet reduces visceral fat and improves metabolic syndrome: A randomized trial.

Overconsumption of carbohydrate-rich food combined with adverse eating patterns contributes to the increasing incidence of metabolic syndrome (MetS) in China. Therefore, we conducted a randomized trial to determine the effects of a low-carbohydrate diet (LCD), an 8-h time-restricted eating (TRE) schedule, and their combination on body weight and abdominal fat area (i.e., primary outcomes) and cardiometabolic outcomes in participants with MetS. Compared with baseline, all 3-month treatments significantly reduce body weight and subcutaneous fat area, but only TRE and combination treatment reduce visceral fat area (VFA), fasting blood glucose, uric acid (UA), and dyslipidemia. Furthermore, compared with changes of LCD, TRE and combination treatment further decrease body weight and VFA, while only combination treatment yields more benefits on glycemic control, UA, and dyslipidemia. In conclusion, without change of physical activity, an 8-h TRE with or without LCD can serve as an effective treatment for MetS (ClinicalTrials.gov: NCT04475822).

A Functional Genomics View of Gibberellin Metabolism in the Cnidarian Symbiont Breviolum minutum.

Dinoflagellate inhabitants of the reef-building corals exchange nutrients and signals with host cells, which often benefit the growth of both partners. Phytohormones serve as central hubs for signal integration between symbiotic microbes and their hosts, allowing appropriate modulation of plant growth and defense in response to various stresses. However, the presence and function of phytohormones in photosynthetic dinoflagellates and their function in the holobionts remain elusive. We hypothesized that endosymbiotic dinoflagellates may produce and employ phytohormones for stress responses. Using the endosymbiont of reef corals Breviolum minutum as model, this study aims to exam whether the alga employ analogous signaling systems by an integrated multiomics approach. We show that key gibberellin (GA) biosynthetic genes are widely present in the genomes of the selected dinoflagellate algae. The non-13-hydroxylation pathway is the predominant route for GA biosynthesis and the multifunctional GA dioxygenase in B. minutum has distinct substrate preference from high plants. GA biosynthesis is modulated by the investigated bleaching-stimulating stresses at both transcriptional and metabolic levels and the exogenously applied GAs improve the thermal tolerance of the dinoflagellate. Our results demonstrate the innate ability of a selected Symbiodiniaceae to produce the important phytohormone and the active involvement of GAs in the coordination and the integration of the stress response.

Widespread Sterol Methyltransferase Participates in the Biosynthesis of Both C4α- and C4ß-Methyl Sterols.

The 4-methyl steranes serve as molecular fossils and are used for studying both eukaryotic evolution and geological history. The occurrence of 4α-methyl steranes in sediments has long been considered evidence of products of partial demethylation mediated by sterol methyl oxidases (SMOs), while 4ß-methyl steranes are attributed entirely to diagenetic generation from 4α-methyl steroids since possible biological sources of their precursor 4ß-methyl sterols are unknown. Here, we report a previously unknown C4-methyl sterol biosynthetic pathway involving a sterol methyltransferase rather than the SMOs. We show that both C4α- and C4ß-methyl sterols are end products of the sterol biosynthetic pathway in an endosymbiont of reef corals, Breviolum minutum, while this mechanism exists not only in dinoflagellates but also in eukaryotes from alveolates, haptophytes, and aschelminthes. Our discovery provides a previously untapped route for the generation of C4-methyl steranes and overturns the paradigm that all 4ß-methyl steranes are diagenetically generated from the 4α isomers. This may facilitate the interpretation of molecular fossils and understanding of the evolution of eukaryotic life in general.

Clade-Specific Sterol Metabolites in Dinoflagellate Endosymbionts Are Associated with Coral Bleaching in Response to Environmental Cues.

Cnidarians cannot synthesize sterols (which play essential roles in growth and development) de novo but often use sterols acquired from endosymbiotic dinoflagellates. While sterol availability can impact the mutualistic interaction between coral host and algal symbiont, the biosynthetic pathways (in the dinoflagellate endosymbionts) and functional roles of sterols in these symbioses are poorly understood. In this study, we found that itraconazole, which perturbs sterol metabolism by inhibiting the sterol 14-demethylase CYP51 in dinoflagellates, induces bleaching of the anemone Heteractis crispa and that bleaching perturbs sterol metabolism of the dinoflagellate. While Symbiodiniaceae have clade-specific sterol metabolites, they share features of the common sterol biosynthetic pathway but with distinct architecture and substrate specificity features of participating enzymes. Tracking sterol profiles and transcripts of enzymes involved in sterol biosynthesis across time in response to different environmental cues revealed similarities and idiosyncratic features of sterol synthesis in the endosymbiont Breviolum minutum Exposure of algal cultures to high levels of light, heat, and acidification led to alterations in sterol synthesis, including blocks through downregulation of squalene synthase transcript levels accompanied by marked growth reductions.IMPORTANCE These results indicate that sterol metabolites in Symbiodiniaceae are clade specific, that their biosynthetic pathways share architectural and substrate specificity features with those of animals and plants, and that environmental stress-specific perturbation of sterol biosynthesis in dinoflagellates can impair a key mutualistic partnership for healthy reefs.

A nematode sterol C4α-methyltransferase catalyzes a new methylation reaction responsible for sterol diversity.

Primitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and Δ7 desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.-.

Transcriptomic and proteomic responses to very low CO2 suggest multiple carbon concentrating mechanisms in Nannochloropsis oceanica.

BACKGROUND: In industrial oleaginous microalgae such as Nannochloropsis spp., the key components of the carbon concentration mechanism (CCM) machineries are poorly defined, and how they are mobilized to facilitate cellular utilization of inorganic carbon remains elusive. RESULTS: For Nannochloropsis oceanica, to unravel genes specifically induced by CO2 depletion which are thus potentially underpinning its CCMs, transcriptome, proteome and metabolome profiles were tracked over 0 h, 3 h, 6 h, 12 h and 24 h during cellular response from high CO2 level (HC; 50,000 ppm) to very low CO2 (VLC; 100 ppm). The activity of a biophysical CCM is evidenced based on induction of transcripts encoding a bicarbonate transporter and two carbonic anhydrases under VLC. Moreover, the presence of a potential biochemical CCM is supported by the upregulation of a number of key C4-like pathway enzymes in both protein abundance and enzymatic activity under VLC, consistent with a mitochondria-implicated C4-based CCM. Furthermore, a basal CCM underpinned by VLC-induced upregulation of photorespiration and downregulation of ornithine-citrulline shuttle and the ornithine urea cycles is likely present, which may be responsible for efficient recycling of mitochondrial CO2 for chloroplastic carbon fixation. CONCLUSIONS: Nannochloropsis oceanica appears to mobilize a comprehensive set of CCMs in response to very low CO2. Its genes induced by the stress are quite distinct from those of Chlamydomonas reinhardtii and Phaeodactylum tricornutum, suggesting tightly regulated yet rather unique CCMs. These findings can serve the first step toward rational engineering of the CCMs for enhanced carbon fixation and biomass productivity in industrial microalgae.

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri.

Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8-Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a "switch" between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8-Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity.

Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii.

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆8,14-sterol (<95%). The combination of [2H3-methyl]methionine incubation to intact cultures showing C28-ergosterol incorporates 2-2H atoms and C29-7-dehydroporiferasterol incorporates 5 2H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ∆24(28)- and ∆25(27)-olefin pathways to C28- and C29-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C28/C29-∆5,7-sterols, viable encysted cells (mature cyst) possess mostly C29-∆5-sterol and non-viable encysted cells possess C28/C29-∆5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ∆5,7-sterol biosynthesis (to ∆5,7) - metabolism (to ∆5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.

Phytosterol Composition of Arachis hypogaea Seeds from Different Maturity Classes.

The seeds of cultivated peanut, Arachis hypogaea, are an agronomically important crop produced for human nutrition, oilseed and feed stock. Peanut seed is the single most expensive variable input cost and thus producers require seed with excellent performance in terms of germination efficiency. During the maturation process, triglycerides are stored in oil bodies as an energy resource during germination and seedling development. The stability of oil body membranes is essential for nutrient mobilization during germination. This study focused on evaluating the phytosterol composition in seed components including the kernel, embryo (heart), and seed coat or skin. Samples of different maturity classes were analyzed for macronutrient and phytosterol content. The three biosynthetic end products in the phytosterol pathway, ß-sitosterol, campesterol and stigmasterol, comprised 82.29%, 86.39% and 94.25% of seed hearts, kernels and seed coats, respectively. Stigmasterol concentration was highest in the seed kernel, providing an excellent source of this sterol known to have beneficial effects on human health. Peanut hearts contained the highest concentration of sterols by mass, potentially providing protection and resources for the developing seedling. The amount of α-tocopherol increases in peanut hearts during the maturation process, providing protection from temperature stress, as well as stability required for seedling vigor. These results suggest that phytosterols may play a significant role in the performance of seeds, and provide a possible explanation for the poor germination efficiency of immature seeds.

CYP51 is an essential drug target for the treatment of primary amoebic meningoencephalitis (PAM).

Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered "rare" (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug 'repurposing'-a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially 'druggable' target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design.

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C28- and C29-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC50 values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼Ki 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.

Inhibition of Arabidopsis chloroplast ß-amylase BAM3 by maltotriose suggests a mechanism for the control of transitory leaf starch mobilisation.

Starch breakdown in leaves at night is tightly matched to the duration of the dark period, but the mechanism by which this regulation is achieved is unknown. In Arabidopsis chloroplasts, ß-amylase BAM3 hydrolyses transitory starch, producing maltose and residual maltotriose. The aim of the current research was to investigate the regulatory and kinetic properties of BAM3. The BAM3 protein was expressed in Escherichia coli and first assayed using a model substrate. Enzyme activity was stimulated by treatment with dithiothreitol and was increased 40% by 2-10 µM Ca2+ but did not require Mg2+. In order to investigate substrate specificity and possible regulatory effects of glucans, we developed a GC-MS method to assay reaction products. BAM3 readily hydrolysed maltohexaose with a Km of 1.7 mM and Kcat of 4300 s-1 but activity was 3.4-fold lower with maltopentaose and was negligible with maltotetraose. With maltohexaose or amylopectin as substrates and using [UL-13C12]maltose in an isotopic dilution method, we discovered that BAM3 activity is inhibited by maltotriose at physiological (mM) concentrations, but not by maltose. In contrast, the extracellular ß-amylase of barley is only weakly inhibited by maltotriose. Our results may explain the impaired starch breakdown in maltotriose-accumulating mutants such as dpe1 which lacks the chloroplast disproportionating enzyme (DPE1) metabolising maltotriose to glucose. We hypothesise that the rate of starch breakdown in leaves can be regulated by inhibition of BAM3 by maltotriose, the concentration of which is determined by DPE, which is in turn influenced by the stromal concentration of glucose. Since the plastid glucose transporter pGlcT catalyses facilitated diffusion between stroma and cytosol, changes in consumption of glucose in the cytosol are expected to lead to concomitant changes in plastid glucose and maltotriose, and hence compensatory changes in BAM3 activity.

Sterols indicate water quality and wastewater treatment efficiency.

As the world's population continues to grow, water pollution is presenting one of the biggest challenges worldwide. More wastewater is being generated and the demand for clean water is increasing. To ensure the safety and health of humans and the environment, highly efficient wastewater treatment systems, and a reliable assessment of water quality and pollutants are required. The advance of holistic approaches to water quality management and the increasing use of ecological water treatment technologies, such as constructed wetlands and waste stabilisation ponds (WSPs), challenge the appropriateness of commonly used water quality indicators. Instead, additional indicators, which are direct measures of the processes involved in the stabilisation of human waste, have to be established to provide an in-depth understanding of system performance. In this study we identified the sterol composition of wastewater treated in WSPs and assessed the suitability of human sterol levels as a bioindicator of treatment efficiency of wastewater in WSPs. As treatment progressed in WSPs, the relative abundance of human faecal sterols, such as coprostanol, epicoprostanol, 24-ethylcoprostanol, and sitostanol decreased significantly and the sterol composition in wastewater changed significantly. Furthermore, sterol levels were found to be correlated with commonly used wastewater quality indicators, such as BOD, TSS and E. coli. Three of the seven sterol ratios that have previously been used to track sewage pollution in the environment, detected a faecal signal in the effluent of WSPs, however, the others were influenced by high prevalence of sterols originating from algal and fungal activities. This finding poses a concern for environmental assessment studies, because environmental pollution from waste stabilisation ponds can go unnoticed. In conclusion, faecal sterols and their ratios can be used as reliable indicators of treatment efficiency and water quality during wastewater treatment in WSPs. They can complement the use of commonly used indicators of water quality, to provide essential information on the overall performance of ponds and whether a pond is underperforming in terms of stabilising human waste. Such a holistic understanding is essential when the aim is to improve the performance of a treatment plant, build new plants or expand existing infrastructure. Future work should aim at further establishing the use of sterols as reliable water quality indicators on a broader scale across natural and engineered systems.

A systems biology approach toward understanding seed composition in soybean.

BACKGROUND: The molecular, biochemical, and genetic mechanisms that regulate the complex metabolic network of soybean seed development determine the ultimate balance of protein, lipid, and carbohydrate stored in the mature seed. Many of the genes and metabolites that participate in seed metabolism are unknown or poorly defined; even more remains to be understood about the regulation of their metabolic networks. A global omics analysis can provide insights into the regulation of seed metabolism, even without a priori assumptions about the structure of these networks. RESULTS: With the future goal of predictive biology in mind, we have combined metabolomics, transcriptomics, and metabolic flux technologies to reveal the global developmental and metabolic networks that determine the structure and composition of the mature soybean seed. We have coupled this global approach with interactive bioinformatics and statistical analyses to gain insights into the biochemical programs that determine soybean seed composition. For this purpose, we used Plant/Eukaryotic and Microbial Metabolomics Systems Resource (PMR, http://www.metnetdb.org/pmr, a platform that incorporates metabolomics data to develop hypotheses concerning the organization and regulation of metabolic networks, and MetNet systems biology tools http://www.metnetdb.org for plant omics data, a framework to enable interactive visualization of metabolic and regulatory networks. CONCLUSIONS: This combination of high-throughput experimental data and bioinformatics analyses has revealed sets of specific genes, genetic perturbations and mechanisms, and metabolic changes that are associated with the developmental variation in soybean seed composition. Researchers can explore these metabolomics and transcriptomics data interactively at PMR.

Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica.

BACKGROUND: Sterols are vital structural and regulatory components in eukaryotic cells; however, their biosynthetic pathways and functional roles in microalgae remain poorly understood. RESULTS: In the oleaginous microalga Nannochloropsis oceanica, the sterol biosynthetic pathway produces phytosterols as minor products and cholesterol as the major product. The evidence together with their deduced biosynthetic pathways suggests that N. oceanica exhibits features of both higher plants and mammals. Temporal tracking of sterol profiles and sterol-biosynthetic transcripts in response to changes in light intensity and nitrogen supply reveal that sterols play roles in cell proliferation, chloroplast differentiation, and photosynthesis. Furthermore, the dynamics of fatty acid (FA) and FA-biosynthetic transcripts upon chemical inhibitor-induced sterol depletion reveal possible co-regulation of sterol production and FA synthesis, in that the squalene epoxidase inhibitor terbinafine reduces sterol content yet significantly elevates free FA production. Thus, a feedback regulation of sterol and FA homeostasis is proposed, with the 1-deoxy-D-xylulose 5-phosphate synthase (DXS, the committed enzyme in isoprenoid and sterol biosynthesis) gene potentially subject to feedback regulation by sterols. CONCLUSION: These findings reveal features of sterol function and biosynthesis in microalgae and suggest new genetic engineering or chemical biology approaches for enhanced oil production in microalgae.