Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov., isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Central Andes in Chile.

Two novel Micromonospora strains, STR1-7T and STR1S-6T, were isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Atacama Desert, Chile. Chemotaxonomic, cultural and phenotypic features confirmed that the isolates belonged to the genus Micromonospora. They grew from 20 to 37 °C, from pH7 to 8 and in the presence of up to 3 %, w/v NaCl. The isolates formed distinct branches in Micromonospora gene trees based on 16S rRNA gene sequences and on a multi-locus sequence analysis of conserved house-keeping genes. A phylogenomic tree generated from the draft genomes of the isolates and their closest phylogenetic neighbours showed that isolate STR1-7T is most closely related to Micromonospora orduensis S2509T, and isolate STR1S-6 T forms a distinct branch that is most closely related to 12 validly named Micromonospora species, including Micromonospora saelicesensis the earliest proposed member of the group. The isolates were separated from one another and from their closest phylogenomic neighbours using a combination of chemotaxonomic, genomic and phenotypic features, and by low average nucleotide index and digital DNA-DNA hybridization values. Consequently, it is proposed that isolates STR1-7T and STR1S-6T be recognized as representing new species in the genus Micromonospora, namely as Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov.; the type strains are STR1-7T (=CECT 9665T=LMG 30768T) and STR1S-6T (=CECT 9666T=LMG 30770T), respectively. Genome mining showed that the isolates have the capacity to produce novel specialized metabolites, notably antibiotics and compounds that promote plant growth, as well as a broad-range of stress-related genes that provide an insight into how they cope with harsh abiotic conditions that prevail in high-altitude Atacama Desert soils.

Coenzyme Q in Thraustochytrium sp. RT2316-16: Effect of the Medium Composition.

Coenzyme Q (CoQ; ubiquinone) is an essential component of the respiratory chain. It is also a potent antioxidant that prevents oxidative damage to DNA, biological membranes, and lipoproteins. CoQ comprises a six-carbon ring with polar substituents that interact with electron acceptors and donors, and a hydrophobic polyisoprenoid chain that allows for its localization in cellular membranes. Human CoQ has 10 isoprenoid units (CoQ10) within the polyisoprenoid chain. Few microorganisms produce CoQ10. This work shows that Thraustochytrium sp. RT2316-16 produces CoQ10 and CoQ9. The CoQ10 content in RT2316-16 depended strongly on the composition of the growth medium and the age of the culture, whereas the CoQ9 content was less variable probably because it served a different function in the cell. Adding p-hydroxybenzoic acid to the culture media positively influenced the CoQ10 content of the cell. The absence of some B vitamins and p-aminobenzoic acid in the culture medium negatively affected the growth of RT2316-16, but reduced the decline in CoQ10 that otherwise occurred during growth. The highest content of CoQ9 and CoQ10 in the biomass were 855 µg g-1 and 10 mg g-1, respectively. The results presented here suggest that the thraustochytrid RT2316-16 can be a potential vehicle for producing CoQ10. Metabolic signals that trigger the synthesis of CoQ10 in RT2316-16 need to be determined for optimizing culture conditions.

Mutactimycin AP, a New Mutactimycin Isolated from an Actinobacteria from the Atacama Desert.

Bacteria belonging to the phylum Actinobacteria are a very good source of antibiotics, and indeed dominate the current clinical antibiotic space. This paper reports Mutactimycin AP, a new compound belonging to an anthracycline-type family of antibiotics, isolated from a Saccharothrix sp. This actinobacterial strain was isolated from the rhizosphere of lupine plants growing in the extreme hyper-arid Atacama Desert. Structural characterization was carried out using electrospray ionization-mass spectrometry (ESI-MS) and NMR spectroscopy in combination with molecular modelling. The compound was tested against the ESKAPE pathogens, where it showed activity against MRSA and five strains associated with bovine mastitis, where it showed activity against Enterococcus pseudoavium and Staphylycoccus Aureus subsp. Aureus.

Generalized Property-Based Encoders and Digital Signal Processing Facilitate Predictive Tasks in Protein Engineering.

Computational methods in protein engineering often require encoding amino acid sequences, i.e., converting them into numeric arrays. Physicochemical properties are a typical choice to define encoders, where we replace each amino acid by its value for a given property. However, what property (or group thereof) is best for a given predictive task remains an open problem. In this work, we generalize property-based encoding strategies to maximize the performance of predictive models in protein engineering. First, combining text mining and unsupervised learning, we partitioned the AAIndex database into eight semantically-consistent groups of properties. We then applied a non-linear PCA within each group to define a single encoder to represent it. Then, in several case studies, we assess the performance of predictive models for protein and peptide function, folding, and biological activity, trained using the proposed encoders and classical methods (One Hot Encoder and TAPE embeddings). Models trained on datasets encoded with our encoders and converted to signals through the Fast Fourier Transform (FFT) increased their precision and reduced their overfitting substantially, outperforming classical approaches in most cases. Finally, we propose a preliminary methodology to create de novo sequences with desired properties. All these results offer simple ways to increase the performance of general and complex predictive tasks in protein engineering without increasing their complexity.

Total mutational load and clinical features as predictors of the metastatic status in lung adenocarcinoma and squamous cell carcinoma patients.

BACKGROUND: Recently, extensive cancer genomic studies have revealed mutational and clinical data of large cohorts of cancer patients. For example, the Pan-Lung Cancer 2016 dataset (part of The Cancer Genome Atlas project), summarises the mutational and clinical profiles of different subtypes of Lung Cancer (LC). Mutational and clinical signatures have been used independently for tumour typification and prediction of metastasis in LC patients. Is it then possible to achieve better typifications and predictions when combining both data streams? METHODS: In a cohort of 1144 Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LSCC) patients, we studied the number of missense mutations (hereafter, the Total Mutational Load TML) and distribution of clinical variables, for different classes of patients. Using the TML and different sets of clinical variables (tumour stage, age, sex, smoking status, and packs of cigarettes smoked per year), we built Random Forest classification models that calculate the likelihood of developing metastasis. RESULTS: We found that LC patients different in age, smoking status, and tumour type had significantly different mean TMLs. Although TML was an informative feature, its effect was secondary to the "tumour stage" feature. However, its contribution to the classification is not redundant with the latter; models trained using both TML and tumour stage performed better than models trained using only one of these variables. We found that models trained in the entire dataset (i.e., without using dimensionality reduction techniques) and without resampling achieved the highest performance, with an F1 score of 0.64 (95%CrI [0.62, 0.66]). CONCLUSIONS: Clinical variables and TML should be considered together when assessing the likelihood of LC patients progressing to metastatic states, as the information these encode is not redundant. Altogether, we provide new evidence of the need for comprehensive diagnostic tools for metastasis.

Production of Carotenoids and Phospholipids by Thraustochytrium sp. in Batch and Repeated-Batch Culture.

The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.

Resilience and sexual health among menopausal women: a cross-sectional study.

OBJECTIVES: Resilience can be defined as the capacity to overcome stressful situations, such as menopausal transition. Female sexual dysfunction is a problem that prevents women from experiencing satisfaction from sexual activity. In this study, we assessed resilience, sexual function, and quality of life among a sample of mid-aged Spanish menopausal women. METHODS: This cross-sectional study was performed in 101 symptomatic menopausal women. Participants filled out the 14-item Wagnild and Young Resilience Scale, the 19-item Female Sexual Function Index (FSFI), the 16-item Cervantes-SF form, and a sociodemographic questionnaire. RESULTS: Resilience scores were significantly higher among women with high sexual function scores (FSFI > 26), with a 14-item Wagnild and Young Resilience Scale total valuation of 86 [80-94] versus 74 [66-79.50] (P<0.001). Sexual function was significantly worse in the low resilience subgroup of participants, with an FSFI total score of 18.90 [14.10-24] versus 29.40 [24.60-33] in the mid-high resilience one (P<0.001). Menopauserelated quality of life was significantly worse among low resilient women, with a Cervantes-SF total score of 42.50 [34.17-56.70] versus 30.80 [20.80-44.90] in the mid-high resilience one (P = 0.004). CONCLUSIONS: In our sample, resilience was related to sexual health. Higher resilience scores seem to be linked to a better quality of life during the menopausal transition.

Nitrogen Sources Affect the Long-Chain Polyunsaturated Fatty Acids Content in Thraustochytrium sp. RT2316-16.

The psychrophilic marine microorganism Thraustochytrium sp. RT2316-16 can produce carotenoids as well as lipids containing the omega-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid and docosahexaenoic acid. This work reports on the effects of the composition of the culture medium, including certain amino acids, on growth and lipid synthesis by RT2316-16. Compared with the culture on glutamate, the use of lysine, alanine, or serine, increased the content of the omega-3 PUFA in total lipids. In the media that contained yeast extract, glutamate, and glucose, lipid accumulation occurred when organic ammonium was exhausted earlier than glucose. In contrast, lipid mobilization was promoted if glucose was exhausted while organic ammonium (supplied by yeast extract and glutamate) remained in the medium. The total content of carotenoids in the lipid-free biomass decreased during the first 12 to 24 h of culture, simultaneously with a decrease in the total lipid content of the biomass. The experimental data suggested a possible interrelationship between the metabolism of carotenoids and lipids. A high content of omega-3 PUFA in the total lipids could be obtained by growing the thraustochytrid in a medium with a low glucose concentration (6 g L-1) and a high concentration of organic nitrogen (yeast extract 12 g L-1; glutamate 1.06 g L-1), after glucose was exhausted. These observations may guide the development of a strategy to enhance omega-3 PUFA in the biomass.

Antarctic Thraustochytrids as Sources of Carotenoids and High-Value Fatty Acids.

Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and carotenoids are needed as human dietary supplements and are essential components in commercial feeds for the production of aquacultured seafood. Microorganisms such as thraustochytrids are potential natural sources of these compounds. This research reports on the lipid and carotenoid production capacity of thraustochytrids that were isolated from coastal waters of Antarctica. Of the 22 isolates, 21 produced lipids containing EPA+DHA, and the amount of these fatty acids exceeded 20% of the total fatty acids in 12 isolates. Ten isolates were shown to produce carotenoids (27.4-63.9 µg/g dry biomass). The isolate RT2316-16, identified as Thraustochytrium sp., was the best producer of biomass (7.2 g/L in five days) rich in carotenoids (63.9 µg/g) and, therefore, became the focus of this investigation. The main carotenoids in RT2316-16 were ß-carotene and canthaxanthin. The content of EPA+DHA in the total lipids (34 ± 3% w/w in dry biomass) depended on the stage of growth of RT2316-16. Lipid and carotenoid content of the biomass and its concentration could be enhanced by modifying the composition of the culture medium. The estimated genome size of RT2316-16 was 44 Mb. Of the 5656 genes predicted from the genome, 4559 were annotated. These included genes of most of the enzymes in the elongation and desaturation pathway of synthesis of ω-3 polyunsaturated fatty acids. Carotenoid precursors in RT2316-16 were synthesized through the mevalonate pathway. A ß-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.

Iron-meditated fungal starvation by lupine rhizosphere-associated and extremotolerant Streptomyces sp. S29 desferrioxamine production.

Siderophores are iron-chelating compounds that aid iron uptake, one of the key strategies for microorganisms to carve out ecological niches in microbially diverse environments. Desferrioxamines are the principal siderophores produced by Streptomyces spp. Their biosynthesis has been well studied and as a consequence, the chemical potential of the pathway continues to expand. With all of this in mind, our study aimed to explore extremotolerant and lupine rhizosphere-derived Streptomyces sp. S29 for its potential antifungal capabilities. Cocultivation of isolate S29 was carried out with Aspergillus niger and Botrytis cinerea, both costly fungal phytopathogens in the wine industry, to simulate their interaction within the rhizosphere. The results indicate that not only is Streptomyces sp. S29 extraordinary at producing hydroxamate siderophores but uses siderophore production as a means to 'starve' the fungi of iron. High resolution LC-MS/MS followed by GNPS molecular networking was used to observe the datasets for desferrioxamines and guided structure elucidation of new desferrioxamine analogues. Comparing the new chemistry, using tools like molecular networking and MS2LDA, with the known biosynthesis, we show that the chemical potential of the desferrioxamine pathway has further room for exploration.

Temperature Differentially Affects Gene Expression in Antarctic Thraustochytrid Oblongichytrium sp. RT2316-13.

Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid's response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.

Cutting the Gordian knot: early and complete amino acid sequence confirmation of class II lasso peptides by HCD fragmentation.

Lasso peptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Their proteolytic and thermal stability alongside their growing potential as therapeutics has increased attention to these antimicrobial peptides. With the advent of genome mining, the discovery of RiPPs allows for the accurate prediction of putatively encoded structures, however, MSn experiments only provide partial sequence confirmation, therefore 2D NMR experiments are necessary for characterisation. Multiple MS/MS techniques were applied to two structurally characterized lasso peptides, huascopeptin and leepeptin, and one uncharacterized lasso peptide, citrulassin C, which was not isolable in sufficient quantity for NMR analysis. We have shown that MS2 can be used to elucidate the full amino acid sequences previously predicted with genome mining for this compound class. HCD was able to open the macrocycles and fragment the newly opened linear peptides, confirming the complete amino acid sequences of the characterised lasso peptides. In addition, to determine if this technique could be applied at the earliest stages of the isolation process, we targeted a lasso peptide found by genome mining, citrulassin C, and were able to fully elucidate the amino acid sequence using only MS2 from a semi-crude extract of Streptomyces huasconensis HST28T.

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13.

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.

Neuropathic pain after thoracotomy: Tracking signs and symptoms before and at monthly intervals following surgery.

BACKGROUND: As the development of neuropathic symptoms contributes to pain severity and chronification after surgery, their early prediction is important to allow targeted treatment. OBJECTIVES: We longitudinally investigated trajectories of signs and symptoms in patients undergoing thoracotomy and assessed whether and at which time they were related to the development of neuropathic pain symptoms 6 months after surgery. METHODS: Presurgical and 6 monthly postsurgical assessments included questionnaires for mental and physical well-being (e.g., depression/anxiety, pain catastrophizing, sleep quality, neuropathic pain symptoms), and quantitative sensory testing (QST). RESULTS: QST trajectories indicated nerve impairment of the surgery site with predominant loss of function. Signs of recovery towards the end of the assessment period were observed for some tests. Unsupervised cluster analysis with NPSI scores 6 months after surgery as clustering variable identified one group with no/low levels of neuropathic symptoms and one with moderate levels. The two groups differed w.r.t. several signs and symptoms already at early time points. Notably, neuropathic pain anywhere in the body differed already preoperatively and sleep impairment differentiated the two groups at all time points. Regression analysis revealed three factors that seemed particularly suited to predicted 6 months NPSI scores, namely preoperative neuropathic pain symptoms, with contributions from sleep impairment 1 month after surgery and the presence of dynamic mechanical allodynia 3 months after surgery. CONCLUSIONS: Clinical routine should focus on the individual's physiological state, including pre-existing neuropathic pain and sleep quality to identify patients early who might be at risk to develop chronic post-surgical neuropathic pain. SIGNIFICANCE: Development of neuropathies contributes to pain severity and pain chronification after surgery. Here we demonstrate trajectories of quantitative sensory tests (assessed at monthly intervals for 6 months after surgery) that reveal accurate time courses of gain/loss of nerve function following thoracotomy. Independent of the degree of neuropathic signs after surgery, the main predictors for post-surgical neuropathic pain are self-reported neuropathic pain before surgery and sleep quality shortly after surgery.

Synthesis of Nß-Substituted α,ß-Diamino Acids via Stereoselective N-Michael Additions to a Chiral Bicyclic Dehydroalanine.

The highly diastereoselective 1,4-conjugate additions of several nitrogen nucleophiles to chiral bicyclic dehydroalanines have been assessed effectively at room temperature in good to excellent yields without needing any catalyst or additional base. This methodology is general, simple, oxygen and moisture tolerant, high-yielding, totally chemo- and stereoselective. This procedure offers an efficient and practical approach for the synthesis of Nß-substituted α,ß-diamino acids, such as 1-isohistidine, τ-histidinoalanine, ß-benzylaminoalanine, ß-(piperidin-1-yl)alanine, ß-(azepan-1-yl)alanine, and fluorescent and ciprofloxacin-containing amino acid derivatives.

Downsizing Class II Lasso Peptides: Genome Mining-Guided Isolation of Huascopeptin Containing the First Gly1-Asp7 Macrocycle.

A new lasso peptide, huascopeptin, was isolated following genome-mined discovery of a new biosynthetic gene cluster in extremotolerant Streptomyces huasconensis HST28T from Salar de Huasco, Atacama Desert, Chile. Compound 1 is a 13-residue class II lasso peptide containing a novel Gly1-Asp7 macrolactam ring, a three-residue loop, and a three-residue tail, making it the smallest lasso peptide isolated to date. The lasso structure was confirmed using NOE restraint-based molecular dynamics simulations.

openPFGE: An open source and low cost pulsed-field gel electrophoresis equipment.

DNA electrophoresis is a fundamental technique in molecular biology that allows the separation of DNA molecules up to ~50 Kbp. Pulsed-field gel electrophoresis [PFGE] is a variation of the conventional DNA electrophoresis technique that allows the separation of very large DNA molecules up to ~10 Mbp. PFGE equipment is very expensive and it becomes an access barrier to many laboratories. Also, just a few privative designs of the equipment are available and it becomes difficult for the community to improve or customize their functioning. Here, we provide an open source PFGE equipment capable of the separation of DNA molecules up to, at least, ~2 Mbp and at low cost: USD$850, about 3% of the price of typical commercial equipment.