Acyl-chain elongation drives ketosynthase substrate selectivity in trans-acyltransferase polyketide synthases

Type I modular polyketide synthases (PKSs), responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl-acyl carrier protein, yielding an extended β-ketoacyl chain. To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. We submitted six KS domains from the trans-acyl transferase PKSs to a mass spectrometry-basedelongation assay, and identified higher substrat selectivity in the elongating step than in preceding acylation. A close correspondence between observed KS selectivity and that predicted by phylogenetic analysis was seen. Our findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates

Complete stereoinversion of L-tryptophan by a fungal single module nonribosomal peptide synthetase

Single-module nonribosomal peptide synthetases (NRPSs) and NRPS-like enzymes activate and transform carboxylic acids in both primary and secondary metabolism and are of great interest due to their biocatalytic potentials. The single-module NRPS IvoA is essential for fungal pigment biosynthesis. Here, we show that IvoA catalyzes ATP-dependent unidirectional stereoinversion of l-tryptophan to d-tryptophan with complete conversion. While the stereoinversion is catalyzed by the epimerization (E) domain, the terminal condensation (C) domain stereoselectively hydrolyzes d-tryptophanyl-S-phosphopantetheine thioester and thus represents a noncanonical C domain function. Using IvoA, we demonstrate a biocatalytic stereoinversion/deracemization route to access a variety of substituted d-tryptophan analogs in high enantiomeric excess

Idiotypic DNA vaccination for the treatment of multiple myeloma: safety and immunogenicity in a phase I clinical study

We report on the safety and immunogenicity of idiotypic DNA vaccination in a phase I, non-randomised, open-label study in patients with multiple myeloma. The study used DNA fusion gene vaccines encoding patient-specific single chain variable fragment, or idiotype (Id), linked to fragment C (FrC) of tetanus toxin. Patients in complete or partial response following high-dose chemotherapy and autologous stem cell transplant were vaccinated intramuscularly with 1 mg DNA on six occasions, beginning at least 6 months post-transplant; follow-up was to week 52. Fourteen patients were enrolled on study and completed vaccinations. Idiotypic DNA vaccines were well tolerated with vaccine-related adverse events limited to low-grade constitutional symptoms. FrC- and Id-specific T-cell responses were detected by ex vivo ELISPOT in 9/14 and 3/14 patients, respectively. A boost of pre-existing anti-FrC antibody (Ab) was detected by ELISA in 8/14 patients, whilst anti-Id Ab was generated in 1/13 patients. Overall, four patients (29 %) made an immune response to FrC and Id, with six patients (43 %) responding to FrC alone. Over the 52-week study period, serum paraprotein was undetectable, decreased or remained stable for ten patients (71 %), whilst ongoing CR/PR was maintained for 11 patients (79 %). The median time to progression was 38.0 months for 13/14 patients. Overall survival was 64 % after a median follow-up of 85.6 months

Eumalacostracan phylogeny and total evidence: limitations of the usual suspects

<p>Abstract</p> <p>Background</p> <p>The phylogeny of Eumalacostraca (Crustacea) remains elusive, despite over a century of interest. Recent morphological and molecular phylogenies appear highly incongruent, but this has not been assessed quantitatively. Moreover, 18S rRNA trees show striking branch length differences between species, accompanied by a conspicuous clustering of taxa with similar branch lengths. Surprisingly, previous research found no rate heterogeneity. Hitherto, no phylogenetic analysis of all major eumalacostracan taxa (orders) has either combined evidence from multiple loci, or combined molecular and morphological evidence.</p> <p>Results</p> <p>We combined evidence from four nuclear ribosomal and mitochondrial loci (18S rRNA, 28S rRNA, 16S rRNA, and cytochrome <it>c </it>oxidase subunit I) with a newly synthesized morphological dataset. We tested the homogeneity of data partitions, both in terms of character congruence and the topological congruence of inferred trees. We also performed Bayesian and parsimony analyses on separate and combined partitions, and tested the contribution of each partition. We tested for potential long-branch attraction (LBA) using taxon deletion experiments, and with relative rate tests. Additionally we searched for molecular polytomies (spurious clades). Lastly, we investigated the phylogenetic stability of taxa, and assessed their impact on inferred relationships over the whole tree. We detected significant conflict between data partitions, especially between morphology and molecules. We found significant rate heterogeneity between species for both the 18S rRNA and combined datasets, introducing the possibility of LBA. As a test case, we showed that LBA probably affected the position of Spelaeogriphacea in the combined molecular evidence analysis. We also demonstrated that several clades, including the previously reported and surprising clade of Amphipoda plus Spelaeogriphacea, are 'supported' by zero length branches. Furthermore we showed that different sets of taxa have the greatest impact upon the relationships within molecular versus morphological trees.</p> <p>Conclusion</p> <p>Rate heterogeneity and conflict between data partitions mean that existing molecular and morphological evidence is unable to resolve a well-supported eumalacostracan phylogeny. We believe that it will be necessary to look beyond the most commonly utilized sources of data (nuclear ribosomal and mitochondrial sequences) to obtain a robust tree in the future.</p

Efficient inference and identifiability analysis for differential equation models with random parameters

Heterogeneity is a dominant factor in the behaviour of many biological processes. Despite this, it is common for mathematical and statistical analyses to ignore biological heterogeneity as a source of variability in experimental data. Therefore, methods for exploring the identifiability of models that explicitly incorporate heterogeneity through variability in model parameters are relatively underdeveloped. We develop a new likelihood-based framework, based on moment matching, for inference and identifiability analysis of differential equation models that capture biological heterogeneity through parameters that vary according to probability distributions. As our novel method is based on an approximate likelihood function, it is highly flexible; we demonstrate identifiability analysis using both a frequentist approach based on profile likelihood, and a Bayesian approach based on Markov-chain Monte Carlo. Through three case studies, we demonstrate our method by providing a didactic guide to inference and identifiability analysis of hyperparameters that relate to the statistical moments of model parameters from independent observed data. Our approach has a computational cost comparable to analysis of models that neglect heterogeneity, a significant improvement over many existing alternatives. We demonstrate how analysis of random parameter models can aid better understanding of the sources of heterogeneity from biological data.Comment: Minor changes to text. Additional results in supplementary material. Additional statistics regarding results given in main and supplementary materia

Direct Measurements of Meltwater Runoff on the Greenland Ice Sheet Surface

Meltwater runoff from the Greenland Ice Sheet surface influences surface mass balance (SMB), ice dynamics and global sea level rise, but is estimated with climate models and thus difficult to validate. We present a way to measure ice surface runoff directly, from hourly in situ supraglacial river discharge measurements and simultaneous high-resolution satellite/drone remote sensing of upstream fluvial catchment area. A first 72-hour trial for a 63.1 square kilometer moulin-terminating internally drained catchment (IDC) on Greenland's mid-elevation (1207-1381 meters above sea level) ablation zone is compared with melt and runoff simulations from HIRHAM5, MAR3.6.1 (Modele Atmospherique Regionale 3.6.1), RACMO2.3 (Regional Atmospheric Climate Model 2.3), MERRA-2 (Modern Era Retrospective-analysis for Research and Applications-2) and SEB climate/SMB models. Current models cannot reproduce peak discharges or timing of runoff entering moulins, but are improved using synthetic unit hydrograph theory (SUH). Retroactive SUH applications to two older field studies reproduces their findings, signifying that remotely sensed IDC area, shape, and river-length are useful for predicting delays in peak runoff delivery to moulins. Applying SUH to HIRHAM5, MAR3.6.1, RACMO2.3 gridded melt products for 799 surrounding IDCs suggests their terminal moulins receive lower peak discharges, less diurnal variability, and asynchronous runoff timing relative to climate/SMB model output alone. Conversely, large IDCs produce high moulin discharges, even at high elevations where melt rates are low. During this particular field experiment models overestimated runoff by plus 21 percent to plus 58 percent, linked to overestimated ablation and possible meltwater retention in bare, low-density ice. Direct measurements of ice surface runoff will improve climate/SMB models, and incorporating remotely sensed IDCs will aid coupling of surface mass balance with ice dynamics and subglacial systems

Mechanism of subunit interaction at ketosynthase-dehydratase junctions in trans-AT polyketide synthases

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products with diverse applications in medicine and agriculture. They typically consist of several multienzyme subunits that utilize structurally-defined docking domains (DDs) at their N- and C-termini to ensure correct assembly into functional multi-protein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-AT modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This involves direct interaction of a largely unstructured docking domain (DD) at the C-terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based cross-linking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene foot-printing and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity