Helmholtz Institute for Pharmaceutical Research Saarland

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Mass spectrometry is a predominant experimental technique in metabolomics and related fields, but metabolite structural elucidation remains highly challenging. We report SIRIUS 4 ( https://bio.informatik.uni-jena.de/sirius/ ), which provides a fast computational approach for molecular structure identification. SIRIUS 4 integrates CSI:FingerID for searching in molecular structure databases. Using SIRIUS 4, we achieved identification rates of more than 70% on challenging metabolomics datasets. SIRIUS 4 is a fast and highly accurate tool for molecular structure interpretation from mass-spectrometry-based metabolomics data.

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.

It is known that extracellular vesicles (EVs) are shed from cells of almost every type of cell or organism, showing their ubiquity in all empires of life. EVs are defined as naturally released particles from cells, delimited by a lipid bilayer, and cannot replicate. These nano- to micrometer scaled spheres shuttle a set of bioactive molecules. EVs are of great interest as vehicles for drug targeting and in fundamental biological research, but in vitro culture of animal cells usually achieves only small yields. The exploration of other biological kingdoms promises comprehensive knowledge on EVs broadening the opportunities for basic understanding and therapeutic use. Thus, plants might be sustainable biofactories producing nontoxic and highly specific nanovectors, whereas bacterial and fungal EVs are promising vaccines for the prevention of infectious diseases. Importantly, EVs from different eukaryotic and prokaryotic kingdoms are involved in many processes including host-pathogen interactions, spreading of resistances, and plant diseases. More extensive knowledge of inter-species and interkingdom regulation could provide advantages for preventing and treating pests and pathogens. In this review, we present a comprehensive overview of EVs derived from eukaryota and prokaryota and we discuss how better understanding of their intercommunication role provides opportunities for both fundamental and applied biology.

The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.

In order to engineer safer nanomaterials, there is a need to understand, systematically evaluate, and develop constructs with appropriate cellular uptake and intracellular fates. The overall goal of this project is to determine the uptake patterns of silica nanoparticle geometries in model cells, in order to aid in the identification of the role of geometry on cellular uptake and transport. In our experiments we observed a significant difference in the viability of two phenotypes of primary macrophages; immortalized macrophages exhibited similar patterns. However, both primary and immortalized epithelial cells did not exhibit toxicity profiles. Interestingly uptake of these geometries in all cell lines exhibited very different time-dependent patterns. A screening of a series of chemical inhibitors of endocytosis was performed to isolate the uptake mechanisms of the different particles. The results show that all geometries exhibit very different uptake profiles and that this may be due to the orientation of the nanoparticles when they interact with the cell surface. Additionally, evidence suggests that these uptake patterns initialize different downstream cellular pathways, dependent on cell type and phenotype.

The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

Plasmids are known to contain genes encoding for virulence factors and antibiotic resistance mechanisms. Their relevance in metagenomic data processing is steadily growing. However, with the increasing popularity and scale of metagenomics experiments, the number of reported plasmids is rapidly growing as well, amassing a considerable number of false positives due to undetected misassembles. Here, our previously published database PLSDB provides a reliable resource for researchers to quickly compare their sequences against selected and annotated previous findings. Within two years, the size of this resource has more than doubled from the initial 13,789 to now 34,513 entries over the course of eight regular data updates. For this update, we aggregated community feedback for major changes to the database featuring new analysis functionality as well as performance, quality, and accessibility improvements. New filtering steps, annotations, and preprocessing of existing records improve the quality of the provided data. Additionally, new features implemented in the web-server ease user interaction and allow for a deeper understanding of custom uploaded sequences, by visualizing similarity information. Lastly, an application programming interface was implemented along with a python library, to allow remote database queries in automated workflows. The latest release of PLSDB is freely accessible under https://www.ccb.uni-saarland.de/plsdb.

Infections with Pseudomonas aeruginosa have become a concerning threat in hospital-acquired infections and for cystic fibrosis patients. The major problem leading to high mortality lies in the appearance of drug-resistant strains. Therefore, a vast number of approaches to develop novel anti-infectives is currently pursued. These diverse strategies span from killing (new antibiotics) to disarming (antivirulence) the pathogen. Particular emphasis lies on the development of compounds that inhibit biofilms formed in chronic infections to restore susceptibility toward antibiotics. Numerous promising results are summarized in this perspective. Antibiotics with a novel mode of action will be needed to avoid cross resistance against currently used therapeutic agents. Importantly, antivirulence drugs are expected to yield a significantly reduced rate of resistance development. Most developments are still far from the application. It can however be expected that combination therapies, also containing antivirulence agents, will pave the way toward novel treatment options against P. aeruginosa.

Covering: 2011-July 2016Myxobacteria are a rich source for structurally diverse secondary metabolites with intriguing biological activities. Here we report on new natural products that were isolated from myxobacteria in the period of 2011 to July 2016. Some examples of recent advances on modes-of-action are also summarised along with a more detailed overview on five compound classes currently assessed in preclinical studies.

Covering: 2013 to June 2018 Heterologous expression of natural product biosynthetic pathways is of increasing interest in microbial biotechnology, drug discovery and optimization. It empowers not only the robust production of valuable biomolecules in more amenable heterologous hosts but also permits the generation of novel analogs through biosynthetic engineering. This strategy also facilitates the discovery of novel bioactive compounds following the functional expression of cryptic biosynthetic gene clusters (BGCs) from fastidious original producers or metagenomic DNA in surrogate hosts, thus facilitating genome mining in the post-genomic era. This review discusses recent advances and trends pertaining to the heterologous production of bacterial natural products, with an emphasis on new techniques, heterologous hosts, and novel chemistry since 2013.

In this review, a general introduction to fragment-based drug design and the underlying concepts is given. General considerations and methodologies ranging from library selection/construction over biophysical screening and evaluation methods to in-depth hit qualification and subsequent optimization strategies are discussed. These principles can be generally applied to most classes of drug targets. The examples given for fragment growing, merging, and linking strategies at the end of the review are set in the fields of enzyme-inhibitor design and macromolecule-macromolecule interaction inhibition. Building upon the foundation of fragment-based drug discovery (FBDD) and its methodologies, we also highlight a few new trends in FBDD.

Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed.

While epithelial cell culture models (e.g., Caco-2 cell line) are widely used to assess the absorption of drug molecules across healthy intestinal mucosa, there are no suitable in vitro models of the intestinal barrier in the state of inflammation. Thus development of novel drugs and formulations for the treatment of inflammatory bowel disease is largely bound to animal models. We here report on the development of a complex in vitro model of the inflamed intestinal mucosa, starting with the selection of suitable enterocyte cell line and proinflammatory stimulus and progressing to the setup and characterization of a three-dimensional coculture of human intestinal epithelial cells and immunocompetent macrophages and dendritic cells. In the 3D setup, controlled inflammation can be induced allowing the mimicking of pathophysiological changes occurring in vivo in the inflamed intestine. Different combinations of proinflammatory stimuli (lipopolysaccharides from Escherichia coli and Salmonella typhimurium, interleukin-1β, interferon-γ) and intestinal epithelial cell lines (Caco-2, HT-29, T84) were evaluated, and only Caco-2 cells were responsive to stimulation, with interleukin-1β being the strongest stimulator. Caco-2 cells responded to the proinflammatory stimulus with a moderate upregulation of proinflammatory markers and a slight, but significant, decrease (20%) of transepithelial electrical resistance (TEER) indicating changes in the epithelial barrier properties. Setting up the coculture model, macrophages and dendritic cells derived from periphery blood monocytes were embedded in a collagen layer on a Transwell filter insert and Caco-2 cells were seeded atop. Even in the presence of immunocompetent cells Caco-2 cells formed a tight monolayer. Addition of IL-1β increased inflammatory cytokine response more strongly compared to Caco-2 single culture and stimulated immunocompetent cells proved to be highly active in sampling apically applied nanoparticles. Thus the 3D coculture provides additional complexity and information compared to the stimulated single cell model. The coculture system may serve as a valuable tool for developing drugs and formulations for the treatment of inflammatory bowel diseases, as well as for studying the interaction of xenobiotics and nanoparticles with the intestinal epithelial barrier in the state of inflammation.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

OBJECTIVES: The liver contains large amounts of microRNA-122 (miR-122), whereas other tissues contain only marginal amounts of this miRNA. MicroRNAs have also been found to circulate in the blood in a cell-free form; their potential as readily accessible disease markers is currently evaluated. Here, we investigated if the serum levels of miR-122 might be useful as disease parameter in patients with chronic hepatitis C virus (HCV) infection. METHODS: RNA was extracted from sera of patients with chronic HCV infection (CHC) and healthy controls and was analyzed for miR-22 content by quantitative real-time reverse-transcription polymerase chain reaction. miR-122 serum levels were correlated with standard parameters of liver function. Liver biopsies from the same patients were examined for the histologic activity index (HAI) and the degree of fibrosis. RESULTS: Sera from patients with CHC contained higher levels of miR-122 than sera from healthy controls. Serum miR-122 levels correlated well with markers of liver inflammatory activity, that is, the serum levels of alanine leucine transaminase (ALT) and aspartate transaminase, and the HAI score. In patients with persistently normal ALT levels, serum miR-122 levels did not differ from healthy controls. There was no correlation of serum miR-122 levels with serum albumin, international normalized ratio, liver fibrosis, or serum HCV RNA. CONCLUSIONS: The serum level of miR-122 strongly correlates with serum ALT activity and with necroinflammatory activity in patients with CHC and elevated ALT levels, but not with fibrosis stage and functional capacity of the liver.

Simultaneous encapsulation of multiple active substances in a single carrier is essential for therapeutic applications of synergistic combinations of drugs. However, traditional carrier systems often lack efficient encapsulation and release of incorporated substances, particularly when combinations of drugs must be released in concentrations of a prescribed ratio. We present a novel biodegradable core-shell carrier system fabricated in a one-step, solvent-free process on a microfluidic chip; a hydrophilic active (doxorubicin hydrochloride) is encapsulated in the aqueous core, while a hydrophobic active (paclitaxel) is encapsulated in the solid shell. Particle size and composition can be precisely controlled, and core and shell can be individually loaded with very high efficiency. Drug-loaded particles can be dried and stored as a powder. We demonstrate the efficacy of this system through the simultaneous encapsulation and controlled release of two synergistic anticancer drugs using two cancer-derived cell lines. This solvent-free platform technology is also of high potential value for encapsulation of other active ingredients and chemical reagents.

MicroRNAs (miRNAs) play indispensable roles in posttranscriptional gene regulation. Their cellular regulatory impact is determined not solely by their sheer number, which likely amounts to >2000 individual miRNAs in human, than by the regulatory effectiveness of single miRNAs. Although, one begins to develop an understanding of the complex mechanisms underlying miRNA-target interactions (MTIs), the overall knowledge of MTI functionality is still rather patchy. In this critical review, we summarize key features of mammalian MTIs. We especially highlight latest insights on (i) the dynamic make-up of miRNA binding sites including non-canonical binding sites, (ii) the cooperativity between miRNA binding sites, (iii) the adaptivity of MTIs through sequence modifications, (iv) the bearing of intra-cellular miRNA localization changes and (v) the role of cell type and cell status specific miRNA interaction partners. The MTI biology is discussed against the background of state-of-the-art approaches with particular emphasis on experimental strategies for evaluating miRNA functionality.