Discretization of Non-uniform Rational B-Spline (NURBS) Models for Meshless Isogeometric Analysis.

We present an algorithm for fast generation of quasi-uniform and variable-spacing nodes on domains whose boundaries are represented as computer-aided design (CAD) models, more specifically non-uniform rational B-splines (NURBS). This new algorithm enables the solution of partial differential equations within the volumes enclosed by these CAD models using (collocation-based) meshless numerical discretizations. Our hierarchical algorithm first generates quasi-uniform node sets directly on the NURBS surfaces representing the domain boundary, then uses the NURBS representation in conjunction with the surface nodes to generate nodes within the volume enclosed by the NURBS surface. We provide evidence for the quality of these node sets by analyzing them in terms of local regularity and separation distances. Finally, we demonstrate that these node sets are well-suited (both in terms of accuracy and numerical stability) for meshless radial basis function generated finite differences discretizations of the Poisson, Navier-Cauchy, and heat equations. Our algorithm constitutes an important step in bridging the field of node generation for meshless discretizations with isogeometric analysis.

Proteomic data and structure analysis combined reveal interplay of structural rigidity and flexibility on selectivity of cysteine cathepsins.

Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided.

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus: Pushing the Limits of Nanopore Sequencing.

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.

Aerobic Utilization of Methanol for Microbial Growth and Production.

Methanol is a reduced one-carbon (C1) compound. It supports growth of aerobic methylotrophs that gain ATP from reduced redox equivalents by respiratory phosphorylation in their electron transport chains. Notably, linear oxidation of methanol to carbon dioxide may yield three reduced redox equivalents if methanol oxidation is NAD-dependent as, e.g., in Bacillus methanolicus. Methanol has a higher degree of reduction per carbon than glucose (6 vs. 4), and thus, lends itself as an ideal carbon source for microbial production of reduced target compounds. However, C-C bond formation in the RuMP or serine cycle, a prerequisite for production of larger molecules, requires ATP and/or reduced redox equivalents. Moreover, heat dissipation and a high demand for oxygen during catabolic oxidation of methanol may pose challenges for fermentation processes. In this chapter, we summarize metabolic pathways for aerobic methanol utilization, aerobic methylotrophs as industrial production hosts, strain engineering, and methanol bioreactor processes. In addition, we provide technological and market outlooks.

Synthetic biology approaches to actinomycete strain improvement.

Their biochemical versatility and biotechnological importance make actinomycete bacteria attractive targets for ambitious genetic engineering using the toolkit of synthetic biology. But their complex biology also poses unique challenges. This mini review discusses some of the recent advances in synthetic biology approaches from an actinomycete perspective and presents examples of their application to the rational improvement of industrially relevant strains.

Metabolic engineering of Amycolatopsis japonicum for optimized production of [S,S]-EDDS, a biodegradable chelator.

The actinomycete Amycolatopsis japonicum is the producer of the chelating compound [S,S]-ethylenediamine-disuccinc acid (EDDS). [S,S]-EDDS is an isomer of ethylenediamine-tetraacetic acid (EDTA), an economically important chelating compound that suffers from an extremely poor degradability. Frequent use of the persistent EDTA in various industrial and domestic applications has caused an accumulation of EDTA in soil as well as in aqueous environments. As a consequence, EDTA is the highest concentrated anthropogenic compound present in water reservoirs. The [S,S]-form of EDDS has chelating properties similar to EDTA, however, in contrast to EDTA it is readily biodegradable. In order to compete with the cost-effective chemical synthesis of EDTA, we aimed to optimize the biotechnological production of [S,S]-EDDS in A. japonicum by using metabolic engineering approaches. Firstly, we integrated several copies of the [S,S]-EDDS biosynthetic genes into the chromosome of A. japonicum and replaced the native zinc responsive promoter with the strong synthetic constitutive promoter SP44*. Secondly, we increased the supply of O-phospho-serine, the direct precursor of [S,S]-EDDS. The combination of these approaches together with the optimized fermentation process led to a significant improvement in [S,S]-EDDS up to 9.8 g/L with a production rate of 4.3 mg/h/g DCW.

A model for potential non-contact ski injuries of the knee.

Broadly accepted is that most knee injuries result from increased vertical forces, usually induced by an incidental ski fall, collision, or a high jump. We present a new non-contact knee injury mechanism that can happen during a ski turn. Such an injury is governed by a sudden inward turn of the inner ski and consequent swing of the inner leg followed by a nearly instant stop when locked by hip and knee joints. The model provides predictive results for a lateral tibial plateau compression fracture because several simplifications have been made. We confirmed that the modelled compression stresses at typical skiing conditions and with typical skiing equipment can provoke serious knee injuries. The awareness of skiers and skiing equipment industry of the described knee injury mechanism can act as an important injury-prevention factor.

Lower ototoxicity and absence of hidden hearing loss point to gentamicin C1a and apramycin as promising antibiotics for clinical use.

Spread of antimicrobial resistance and shortage of novel antibiotics have led to an urgent need for new antibacterials. Although aminoglycoside antibiotics (AGs) are very potent anti-infectives, their use is largely restricted due to serious side-effects, mainly nephrotoxicity and ototoxicity. We evaluated the ototoxicity of various AGs selected from a larger set of AGs on the basis of their strong antibacterial activities against multidrug-resistant clinical isolates of the ESKAPE panel: gentamicin, gentamicin C1a, apramycin, paromomycin and neomycin. Following local round window application, dose-dependent effects of AGs on outer hair cell survival and compound action potentials showed gentamicin C1a and apramycin as the least toxic. Strikingly, although no changes were observed in compound action potential thresholds and outer hair cell survival following treatment with low concentrations of neomycin, gentamicin and paromomycin, the number of inner hair cell synaptic ribbons and the compound action potential amplitudes were reduced. This indication of hidden hearing loss was not observed with gentamicin C1a or apramycin at such concentrations. These findings identify the inner hair cells as the most vulnerable element to AG treatment, indicating that gentamicin C1a and apramycin are promising bases for the development of clinically useful antibiotics.

Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency.

Coenzyme A is an essential metabolite known for its central role in over one hundred cellular metabolic reactions. In cells, Coenzyme A is synthesized de novo in five enzymatic steps with vitamin B5 as the starting metabolite, phosphorylated by pantothenate kinase. Mutations in the pantothenate kinase 2 gene cause a severe form of neurodegeneration for which no treatment is available. One therapeutic strategy is to generate Coenzyme A precursors downstream of the defective step in the pathway. Here we describe the synthesis, characteristics and in vivo rescue potential of the acetyl-Coenzyme A precursor S-acetyl-4'-phosphopantetheine as a possible treatment for neurodegeneration associated with pantothenate kinase deficiency.

Generation of Lactobacillus plantarum strains with improved potential to target gastrointestinal disorders related to sugar malabsorption.

Malabsorption of dietary sugars is a common cause of gastrointestinal discomfort, affecting up to one in three people with debilitating symptoms, such as abdominal pain, osmotic diarrhoea, bloating and flatulence. Besides dietary interventions, it has been suggested that ingestion of lactobacilli may alleviate these symptoms. The objectives of this study were to generate strains with improved potential to ameliorate sugar malabsorption related gastrointestinal disorders. Initial selection was made from 183 natural isolates of lactic acid bacteria, on the basis of broad sugar fermentation ability, absence of gas production, gastrointestinal survival and susceptibility to important medical antimicrobials. Two strains of L. plantarum (KR6 and M5) exhibited favourable characteristics for all criteria, and were further optimised through random mutagenesis and selection approaches. Ultraviolet light (UV) exposure resulted in mutants characterized by better survival (for 1.9 log and 1.4 log) in gastrointestinal conditions. Subsequent exposure to ethyl methanesulfonate (EMS) provided mutants with greater tolerance to glucose induced catabolic repression. UV and UV-EMS mutants of L. plantarum M5 showed improved adhesion ability. As a result of this optimisation, L. plantarum MP2026 and L. plantarum MP2420 have been identified as promising candidates for probiotics, intended for alleviation of gastrointestinal discomfort originating from sugar malabsorption.

Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea.

BACKGROUND: Omics approaches have significantly increased our understanding of biological systems. However, they have had limited success in explaining the dramatically increased productivity of commercially important natural products by industrial high-producing strains, such as the erythromycin-producing actinomycete Saccharopolyspora erythraea. Further yield increase is of great importance but requires a better understanding of the underlying physiological processes. RESULTS: To reveal the mechanisms related to erythromycin yield increase, we have undertaken an integrated study of the genomic, transcriptomic, and proteomic differences between the wild type strain NRRL2338 (WT) and the industrial high-producing strain ABE1441 (HP) of S. erythraea at multiple time points of a simulated industrial bioprocess. 165 observed mutations lead to differences in gene expression profiles and protein abundance between the two strains, which were most prominent in the initial stages of erythromycin production. Enzymes involved in erythromycin biosynthesis, metabolism of branched chain amino acids and proteolysis were most strongly upregulated in the HP strain. Interestingly, genes related to TCA cycle and DNA-repair were downregulated. Additionally, comprehensive data analysis uncovered significant correlations in expression profiles of the erythromycin-biosynthetic genes, other biosynthetic gene clusters and previously unidentified putative regulatory genes. Based on this information, we demonstrated that overexpression of several genes involved in amino acid metabolism can contribute to increased yield of erythromycin, confirming the validity of our systems biology approach. CONCLUSIONS: Our comprehensive omics approach, carried out in industrially relevant conditions, enabled the identification of key pathways affecting erythromycin yield and suggests strategies for rapid increase in the production of secondary metabolites in industrial environment.

On the Convergence of Stresses in Fretting Fatigue.

Fretting is a phenomenon that occurs at the contacts of surfaces that are subjected to oscillatory relative movement of small amplitudes. Depending on service conditions, fretting may significantly reduce the service life of a component due to fretting fatigue. In this regard, the analysis of stresses at contact is of great importance for predicting the lifetime of components. However, due to the complexity of the fretting phenomenon, analytical solutions are available for very selective situations and finite element (FE) analysis has become an attractive tool to evaluate stresses and to study fretting problems. Recent laboratory studies in fretting fatigue suggested the presence of stress singularities in the stick-slip zone. In this paper, we constructed finite element models, with different element sizes, in order to verify the existence of stress singularity under fretting conditions. Based on our results, we did not find any singularity for the considered loading conditions and coefficients of friction. Since no singularity was found, the present paper also provides some comments regarding the convergence rate. Our analyses showed that the convergence rate in stress components depends on coefficient of friction, implying that this rate also depends on the loading condition. It was also observed that errors can be relatively high for cases with a high coefficient of friction, suggesting the importance of mesh refinement in these situations. Although the accuracy of the FE analysis is very important for satisfactory predictions, most of the studies in the literature rarely provide information regarding the level of error in simulations. Thus, some recommendations of mesh sizes for those who wish to perform FE analysis of fretting problems are provided for different levels of accuracy.

Roles of the crotonyl-CoA carboxylase/reductase homologues in acetate assimilation and biosynthesis of immunosuppressant FK506 in Streptomyces tsukubaensis.

BACKGROUND: In microorganisms lacking a functional glyoxylate cycle, acetate can be assimilated by alternative pathways of carbon metabolism such as the ethylmalonyl-CoA (EMC) pathway. Among the enzymes converting CoA-esters of the EMC pathway, there is a unique carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase (Ccr). In addition to the EMC pathway, gene homologues of ccr can be found in secondary metabolite gene clusters that are involved in the provision of structurally diverse extender units used in the biosynthesis of polyketide natural products. The roles of multiple ccr homologues in the same genome and their potential interactions in primary and secondary metabolic pathways are poorly understood. RESULTS: In the genome of S. tsukubaensis we have identified two ccr homologues; ccr1 is located in the putative ethylmalonyl-CoA (emc) operon and allR is located on the left fringe of the FK506 cluster. AllR provides an unusual extender unit allylmalonyl-CoA (ALL) for the biosynthesis of FK506 and potentially also ethylmalonyl-CoA for the related compound FK520. We have demonstrated that in S. tsukubaensis the ccr1 gene does not have a significant role in the biosynthesis of FK506 or FK520 when cultivated on carbohydrate-based media. However, when overexpressed under the control of a strong constitutive promoter, ccr1 can take part in the biosynthesis of ethylmalonyl-CoA and thereby FK520, but not FK506. In contrast, if ccr1 is inactivated, allR is not able to sustain a functional ethylmalonyl-CoA pathway (EMC) and cannot support growth on acetate as the sole carbon source, even when constitutively expressed in the chimeric emc operon. This is somewhat surprising considering that the same chimeric emc operon results in production of FK506 as well as FK520, consistent with the previously proposed relaxed specificity of AllR for C4 and C5 substrates. CONCLUSIONS: Different regulation of the expression of both ccr genes, ccr1 and allR, and their corresponding pathways EMC and ALL, respectively, in combination with the different enzymatic properties of the Ccr1 and AllR enzymes, determine an almost exclusive role of ccr1 in the EMC pathway in S. tsukubaensis, and an exclusive role of allR in the biosynthesis of FK506/FK520, thus separating the functional roles of these two genes between the primary and secondary metabolic pathways.

Extracellular 4'-phosphopantetheine is a source for intracellular coenzyme A synthesis.

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Rapid identification of atypical tetracyclines using tandem mass spectrometric fragmentation patterns.

RATIONALE: When applying biosynthetic engineering approaches at the early stages of drug discovery, e.g. aiming to develop novel tetracycline analogues, target compounds are generally produced by engineered microorganisms in low yields. Rapid and reliable identification of metabolites with desired structural modification directly from bacterial cultures is therefore of great importance. METHODS: Structural elucidation of atypical tetracyclines was carried out by fragmentation applying electrospray ionisation tandem mass spectrometry (ESI-MS/MS) (triple quadrupole - linear ion trap; Applied Biosystems 4000 QTRAP) and a high-resolution mass spectrometer (Agilent Technologies 6224 TOF). Fragmentation patterns were obtained either with direct injection or by applying separation of target compounds with high-performance liquid chromatography (HPLC) prior to mass spectrometry. In-source and CID fragmentation were compared. Theoretical calculations of target structures using the Gaussian programme suite were carried out with the aim of strengthening experimental structural elucidation. RESULTS: Recombinant strains of Amycolatopsis sulphurea producing atypical tetracyclines chelocardin, modified chelocardin analogues (9-demethylchelocardin and 2-carboxyamido-2-deacetyl-chelocardin (CDCHD), and anhydrotetracycline (ATC) were analysed by collision-induced dissociation (CID) fragmentation with higher collision energies to yield structurally important fragments which were identified. We have demonstrated that ATC is more prone to fragmentation compared to its epimer, which was further supported by comparison of both structures calculated with ab initio calculations. CONCLUSIONS: We have demonstrated that fragmentation patterns of atypical tetracyclines in CID-MS spectra enable rapid structural elucidation of target metabolites produced by cultures of genetically engineered bacteria. This method is of significant importance for early stages of drug development considering that isolation of target metabolites produced at low concentration is challenging. Copyright © 2015 John Wiley & Sons, Ltd.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

BACKGROUND: Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. RESULTS: We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. CONCLUSIONS: SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.

Draft Genome Sequence of Streptomyces rapamycinicus Strain NRRL 5491, the Producer of the Immunosuppressant Rapamycin.

Streptomyces rapamycinicus strain NRRL 5491 produces the important drug rapamycin. It has a large genome of 12.7 Mb, of which over 3 Mb consists of 48 secondary metabolite biosynthesis clusters.

Draft Genome Sequence of the Oxytetracycline-Producing Bacterium Streptomyces rimosus ATCC 10970.

We report the draft genome of Streptomyces rimosus (ATCC 10970), a soil isolate that produces oxytetracycline, a commercially important and clinically useful antibiotic.

The proinflammatory cytokines interleukin-1α and tumor necrosis factor α promote the expression and secretion of proteolytically active cathepsin S from human chondrocytes.

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.