Core-Shell DNA-Cholesterol Nanoparticles Exert Lysosomolytic Activity in African Trypanosomes.

Trypanosoma brucei is the causal infectious agent of African trypanosomiasis in humans and Nagana in livestock. Both diseases are currently treated with a small number of chemotherapeutics, which are hampered by a variety of limitations reaching from efficacy and toxicity complications to drug-resistance problems. Here, we explore the forward design of a new class of synthetic trypanocides based on nanostructured, core-shell DNA-lipid particles. In aqueous solution, the particles self-assemble into micelle-type structures consisting of a solvent-exposed, hydrophilic DNA shell and a hydrophobic lipid core. DNA-lipid nanoparticles have membrane-adhesive qualities and can permeabilize lipid membranes. We report the synthesis of DNA-cholesterol nanoparticles, which specifically subvert the membrane integrity of the T. brucei lysosome, killing the parasite with nanomolar potencies. Furthermore, we provide an example of the programmability of the nanoparticles. By functionalizing the DNA shell with a spliced leader (SL)-RNA-specific DNAzyme, we target a second trypanosome-specific pathway (dual-target approach). The DNAzyme provides a backup to counteract the recovery of compromised parasites, which reduces the risk of developing drug resistance.

Analyzing editosome function in high-throughput.

Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.

The OB-fold proteins of the Trypanosoma brucei editosome execute RNA-chaperone activity.

Sequence-deficient mitochondrial pre-mRNAs in African trypanosomes are substrates of a U-nucleotide-specific RNA editing reaction to generate translation-competent mRNAs. The reaction is catalyzed by a macromolecular protein complex termed the editosome. Editosomes execute RNA-chaperone activity to overcome the highly folded nature of pre-edited substrate mRNAs. The molecular basis for this activity is unknown. Here we test five of the OB-fold proteins of the Trypanosoma brucei editosome as candidates. We demonstrate that all proteins execute RNA-chaperone activity albeit to different degrees. We further show that the activities correlate to the surface areas of the proteins and we map the protein-induced RNA-structure changes using SHAPE-chemical probing. To provide a structural context for our findings we calculate a coarse-grained model of the editosome. The model has a shell-like structure: Structurally well-defined protein domains are separated from an outer shell of intrinsically disordered protein domains, which suggests a surface-driven mechanism for the chaperone activity.

On-Demand Production of Femtoliter Drops in Microchannels and Their Use as Biological Reaction Compartments.

We present a method allowing to produce monodisperse droplets with volumes in the femtoliter range in a microchannel on demand. The method utilizes pulsed electric fields deforming the interface between an aqueous and an oil phase and pinching off droplets. Water and xanthan gum solutions are considered as disperse-phase liquids, and it is shown that the method can be applied even to solutions with a zero-shear rate viscosity more than 104-times higher than that of water. The droplet formation regimes are explored by systematically varying the pulse amplitude and duration as well as the salt concentration. The dependence of the process on the pulse amplitude can be utilized to tune the droplet size. To demonstrate the applicability of the electric-field-driven droplet generator, it is shown that the droplets can be used as versatile biological reaction compartments. It is proven that droplets containing a cell-free transcription-translation system execute gene transcription and protein biosynthesis in a timely and programmable fashion. Moreover, it is verified that biomolecules inside the aqueous droplets such as small RNAs can be diffusionally activated from the outside to induce a ligand-driven biochemical switch.

Bioinspired Design of Lysolytic Triterpenoid-Peptide Conjugates that Kill African Trypanosomes.

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). The active TLF ingredient is the primate-specific apolipoprotein L1 (APOL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies, only two are able to counteract the activity of APOL1; this illustrates its evolutionarily optimized design and trypanocidal potency. Herein, we ask whether a synthetic (syn) TLF can be synthesized by using the design principles of the natural TLF complexes but with different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell-binding, uptake and lysosomal-transport modules and the synthetic peptide GALA acts as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis thus demonstrating a proof-of-principle for the bioinspired, forward-design of a synTLF.

'Gestalt,' composition and function of the Trypanosoma brucei editosome.

RNA editing describes a chemically diverse set of biomolecular reactions in which the nucleotide sequence of RNA molecules is altered. Editing reactions have been identified in many organisms and frequently contribute to the maturation of organellar transcripts. A special editing reaction has evolved within the mitochondria of the kinetoplastid protozoa. The process is characterized by the insertion and deletion of uridine nucleotides into otherwise nontranslatable messenger RNAs. Kinetoplastid RNA editing involves an exclusive class of small, noncoding RNAs known as guide RNAs. Furthermore, a unique molecular machinery, the editosome, catalyzes the process. Editosomes are megadalton multienzyme assemblies that provide a catalytic surface for the individual steps of the reaction cycle. Here I review the current mechanistic understanding and molecular inventory of kinetoplastid RNA editing and the editosome machinery. Special emphasis is placed on the molecular morphology of the editing complex in order to correlate structural features with functional characteristics.

RNA helicases involved in U-insertion/deletion-type RNA editing.

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa such as the disease-causing African trypanosomes are substrates of a unique RNA editing reaction. The process is characterized by the site-specific insertion and deletion of exclusively U nucleotides and converts nonfunctional pre-mRNAs into translatable transcripts. Similar to other RNA-based metabolic pathways, RNA editing is catalyzed by a macromolecular protein complex, the editosome. Editosomes provide a reactive surface for the individual steps of the catalytic cycle and involve as key players a specific class of small, non-coding RNAs termed guide (g)RNAs. gRNAs basepair proximal to an editing site and act as quasi templates in the U-insertion/deletion reaction. Next to the editosome several accessory proteins and complexes have been identified, which contribute to different steps of the reaction. This includes matchmaking-type RNA/RNA annealing factors as well as RNA helicases of the archetypical DEAD- and DExH/D-box families. Here we summarize the current structural, genetic and biochemical knowledge of the two characterized "editing RNA helicases" and provide an outlook onto dynamic processes within the editing reaction cycle. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Trypanosoma brucei 20 S editosomes have one RNA substrate-binding site and execute RNA unwinding activity.

Editing of mitochondrial pre-mRNAs in African trypanosomes generates full-length transcripts by the site-specific insertion and deletion of uridylate nucleotides. The reaction is catalyzed by a 0.8 MDa multienzyme complex, the editosome. Although the binding of substrate pre-edited mRNAs and cognate guide RNAs (gRNAs) represents the first step in the reaction cycle, the biochemical and biophysical details of the editosome/RNA interaction are not understood. Here we show that editosomes bind full-length substrate mRNAs with nanomolar affinity in a nonselective fashion. The complexes do not discriminate-neither kinetically nor thermodynamically-between different mitochondrial pre-mRNAs or between edited and unedited versions of the same transcript. They also bind gRNAs and gRNA/pre-mRNA hybrid RNAs with similar affinities and association rate constants. Gold labeling of editosome-bound RNA in combination with transmission electron microscopy identified a single RNA-binding site per editosome. However, atomic force microscopy of individual pre-mRNA-editosome complexes revealed that multiple editosomes can interact with one pre-mRNA. Lastly, we demonstrate a so far unknown activity of the editing machinery: editosome-bound RNA becomes unfolded by a chaperone-type RNA unwinding activity.

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo.

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of approximately 20S and approximately 35-40S and present the three-dimensional structures of both complexes by electron microscopy. The approximately 35-40S complexes consist of a platform density packed against a semispherical element. The approximately 20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The approximately 20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Nanopore-Based Direct RNA Sequencing of the Trypanosoma brucei Transcriptome Identifies Novel lncRNAs.

Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay, to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized to date. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known, because of the inherent cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here, we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome Trypanosoma brucei, with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 32 of which are stage-specifically expressed. We also present insights into the complexity of the T. brucei transcriptome, including alternative transcriptional start and stop sites and potential transcript isoforms, to provide a bias-free understanding of the intricate RNA landscape in T. brucei.

Developing a framework for investigating citizen science through a combination of web analytics and social science methods-The CS Track perspective.

Over the past decade, Citizen Science (CS) has shown great potential to transform the power of the crowd into knowledge of societal value. Many projects and initiatives have produced high quality scientific results by mobilizing peoples' interest in science to volunteer for the public good. Few studies have attempted to map citizen science as a field, and assess its impact on science, society and ways to sustain its future practice. To better understand CS activities and characteristics, CS Track employs an analytics and analysis framework for monitoring the citizen science landscape. Within this framework, CS Track collates and processes information from project websites, platforms and social media and generates insights on key issues of concern to the CS community, such as participation patterns or impact on science learning. In this paper, we present the operationalization of the CS Track framework and its three-level analysis approach (micro-meso-macro) for applying analytics techniques to external data sources. We present three case studies investigating the CS landscape using these analytical levels and discuss the strengths and limitations of combining web-analytics with quantitative and qualitative research methods. This framework aims to complement existing methods for evaluating CS, address gaps in current observations of the citizen science landscape and integrate findings from multiple studies and methodologies. Through this work, CS Track intends to contribute to the creation of a measurement and evaluation scheme for CS and improve our understanding about the potential of analytics for the evaluation of CS.

Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

Issues in the assessment of non-inferiority: perspectives drawn from case studies.

Superiority claims for improved efficacy are the backbone of clinical development of new therapies. However, not every new therapy in development allows for such a claim. Some therapies per se do not try to improve efficacy further but concentrate on important aspects in safety or convenience. Such improvements can be equally important to patients, and development strategies should be available for such compounds. A three-arm design with placebo, active control and experimental treatment may be viewed as the golden standard for such compounds; however, it may be difficult if not impossible to add a placebo arm in certain diseases. In such situations, non-inferiority designs are the only development option left. This paper will highlight some of the key issues with such designs in practice and will report experience from two studies from different therapeutic areas intended for regulatory submission.

A Virtual Sandbox Approach to Studying the Effect of Augmented Communication on Human-Robot Collaboration.

Human-Robot Collaboration (HRC) has the potential for a paradigm shift in industrial production by complementing the strengths of industrial robots with human staff. However, exploring these scenarios in physical experimental settings is costly and difficult, e.g., due to safety considerations. We present a virtual reality application that allows the exploration of HRC work arrangements with autonomous robots and their effect on human behavior. Prior experimental studies conducted using this application demonstrated the benefits of augmenting an autonomous robot arm with communication channels on subjective aspects such as perceived stress. Motivated by current safety regulations that hinder HRC to expand its full potential, we explored the effects of the augmented communication on objective measures (collision rate and produced goods) within a virtual sandbox application. Explored through a safe and replicable setup, the goal was to determine whether communication channels that provide guidance and explanation on the robot can help mitigate safety hazards without interfering with the production effectiveness of both parties. This is based on the theoretical foundation that communication channels enable the robot to explain its action, helps the human collaboration partner to comprehend the current state of the shared task better, and react accordingly. Focused on the optimization of production output, reduced collision rate, and increased perception of safety, a between-subjects experimental study with two conditions (augmented communication vs non-augmented) was conducted. The results revealed a statistically significant difference in terms of production quantity output and collisions with the robot, favoring the augmented conditions. Additional statistically significant differences regarding self-reported perceived safety were found. The results of this study provide an entry point for future research regarding the augmentation of industrial robots with communication channels for safety purposes.

Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE).

Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors. Graphic abstract: Characteristics of the fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE) assay.

TbMP42 is a structure-sensitive ribonuclease that likely follows a metal ion catalysis mechanism.

RNA editing in African trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. The process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. While for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. TbMP42, is a component of the editosome that is characterized by two C(2)H(2)-type zinc-finger domains and a putative oligosaccharide/oligonucleotide-binding fold. Recombinant TbMP42 has been shown to possess endo/exoribonuclease activity in vitro; however, the protein lacks canonical nuclease motifs. Using a set of synthetic gRNA/pre-mRNA substrate RNAs, we demonstrate that TbMP42 acts as a topology-dependent ribonuclease that is sensitive to base stacking. We further show that the chelation of Zn(2+) cations is inhibitory to the enzyme activity and that the chemical modification of amino acids known to coordinate Zn(2+) inactivates rTbMP42. Together, the data are suggestive of a Zn(2+)-dependent metal ion catalysis mechanism for the ribonucleolytic activity of rTbMP42.

Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing.

Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8 MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and as a consequence generates a simplified RNA folding landscape that is critical for the progression of the editing reaction cycle. Here we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (double-stranded) versus structurally unconstrained (single-stranded) nucleotides. Electrophilic anhydrides such as 1-methyl-7-nitroisatoic anhydride are used as probing reagents, and the ribose 2'-modified nucleotides are mapped as abortive cDNA synthesis products. As a result, SHAPE allows the identification of all single-stranded and base-paired regions in a given RNA, and the data are used to compute experimentally derived RNA 2D structures. A side-by-side comparison of the RNA 2D folds in the pre- and post-chaperone states finally maps the chaperone-induced dynamic of the different pre-mRNAs with single-nucleotide resolution.

Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes.

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Cell-Free Prototyping of AND-Logic Gates Based on Heterogeneous RNA Activators.

RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages such as short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multilevel regulated circuits. In this work, we combined a variety of small transcriptional activator RNAs (STARs) and toehold switches to build highly effective AND-gates. To characterize the components and their dynamic range, we used an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets. We analyzed a prototype gate in vitro as well as in silico, employing parametrized ordinary differential equations (ODEs), for which parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. On the basis of this analysis, we created nine additional AND-gates and tested them in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, offering a dynamic range comparable to the level of protein circuits. This study shows the potential of a rapid prototyping approach for RNA circuit design, using cell-free systems in combination with a model prediction.

Strategies for atopy prevention.

Allergic diseases, particularly in childhood, have become one of the epidemics of the 21st century. Whereas previous strategies for allergy prevention focused on the avoidance of risk factors, more recent approaches are addressing attempts to provide protective factors to infants and young children to achieve immune modulation and tolerance to harmless nutritional or environmental allergens. This change of paradigm for allergy prevention might lead to more effective interventions, which hopefully contribute to reversing the epidemiologic trend of the last decades. In many industrialized countries, the increased prevalence of atopy and asthma has become a serious public health issue. If preventive intervention could be effective at all, it would have to be applied early in life, most probably in early infancy. Unfortunately, our understanding of the natural history of the process of atopic sensitization, atopic dermatitis, and allergic airway disease is still very limited. On the other hand, the evaluation of risk factors and determinants is a necessary prerequisite for any effective intervention.