Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation.

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

Does fear-of-failure mediate the relationship between educational expectations and stress-related complaints among Swedish adolescents? A structural equation modelling approach.

BACKGROUND: This study investigated the possible mediating role of fear-of-failure between educational expectations and adolescent stress-related complaints with a specific focus on gender differences among Swedish adolescents, and related these findings more broadly to school-related demands and stress-related complaints. METHODS: A total of N = 5504 Swedish adolescents (Mage = 15 years, SD = 0.0 years, 50.2% girls) were drawn from the 2018 Swedish Programme for International Student Assessment study for our investigation. We used structural equation models to explore if fear-of-failure mediates the relationship between educational expectations and negative affect, with a specific focus on gender differences. Educational expectations were utilized in the measurement model. Fear-of-failure was constructed as a latent mediating variable. Negative affect was constructed as a latent variable and utilized as an outcome variable. We subsequently undertook bootstrapping tests of indirect effects and non-linear comparisons of indirect effects to assess the reliability of the results. RESULTS: Fear-of-failure partially mediated the association between educational expectations and negative affect (39%). Our gender-specific structural equation model demonstrated that this relationship was more pronounced for girls, suggesting girls are more vulnerable to negative affect as a result of experiencing higher levels of fear of failing. CONCLUSIONS: The findings suggest that fear-of-failure partially explains the association between educational expectations and negative affect and that this association is more pronounced for girls. This study provides insights into better understanding adolescent stress-related complaints, and the differential role fear of failing has in regards to gender.

Solid Phase Synthesis of DNA Nanostructures in Heavy Liquid.

Introduction of the solid phase method to synthesize biopolymers has revolutionized the field of biological research by enabling efficient production of peptides and oligonucleotides. One of the advantages of this method is the ease of removal of excess production materials from the desired product, as it is immobilized on solid substrate. The DNA origami method utilizes the nature of nucleotide base-pairing to construct well-defined objects at the nanoscale, and has become a potent tool for manipulating matter in the fields of chemistry, physics, and biology. Here, the development of an approach to synthesize DNA nanostructures directly on magnetic beads, where the reaction is performed in heavy liquid to maintain the beads in suspension is reported. It is demonstrated that the method can achieve high folding yields of up to 90% for various DNA shapes, comparable to standard folding. At the same time, this establishes an easy, fast, and efficient way to further functionalize the DNA origami in one-pot, as well as providing a built-in purification method for easy removal of excess by-products such as non-integrated DNA strands and residual functionalization molecules.

Trends in adolescent psychosomatic complaints: a quantile regression analysis of Swedish HBSC data 1985-2017.

BACKGROUND AND AIMS: According to recent criticism, survey-based measures of adolescent psychosomatic complaints have poor content validity insofar as they conflate trivial with severe complaints. It is argued that this means that estimates of prevalence and trends in complaints may reflect trivial complaints that are not indicators of health problems. In this study, two observable implications of this criticism were investigated: (a) that self-reported psychosomatic complaints should have a bimodal distribution; and (b) that the increase in complaints over time should be of approximately equal size throughout the distribution of complaints. METHODS: Three decades (1985/1986-2017/2018) of repeated cross-sectional data from the Swedish Health Behaviour in School-aged Children survey were used. Psychosomatic complaints were measured using the screening instrument Health Behaviour in School-aged Children symptom checklist. Histograms, bar charts and quantile regression models were used for the analysis. RESULTS AND CONCLUSIONS: With regard to the first implication, the results showed that the distribution of complaints was not bimodal and that there were no clusters of respondents. This suggests that binary categorisations of students can be reductive and conceal important variations across students. With regard to the second implication, the results showed that the increase in complaints was greatest among students who report frequent and co-occurring complaints. This suggests that reports of increasing complaints in adolescents cannot be explained as being primarily due to a greater inclination to report trivial complaints. It is concluded that any conflation of trivial and more severe complaints in surveys of psychosomatic complaints is not reflected in population-based estimates.

Wireframe DNA Origami for the Cellular Delivery of Platinum(II)-Based Drugs.

The DNA origami method has revolutionized the field of DNA nanotechnology since its introduction. These nanostructures, with their customizable shape and size, addressability, nontoxicity, and capacity to carry bioactive molecules, are promising vehicles for therapeutic delivery. Different approaches have been developed for manipulating and folding DNA origami, resulting in compact lattice-based and wireframe designs. Platinum-based complexes, such as cisplatin and phenanthriplatin, have gained attention for their potential in cancer and antiviral treatments. Phenanthriplatin, in particular, has shown significant antitumor properties by binding to DNA at a single site and inhibiting transcription. The present work aims to study wireframe DNA origami nanostructures as possible carriers for platinum compounds in cancer therapy, employing both cisplatin and phenanthriplatin as model compounds. This research explores the assembly, platinum loading capacity, stability, and modulation of cytotoxicity in cancer cell lines. The findings indicate that nanomolar quantities of the ball-like origami nanostructure, obtained in the presence of phenanthriplatin and therefore loaded with that specific drug, reduced cell viability in MCF-7 (cisplatin-resistant breast adenocarcinoma cell line) to 33%, while being ineffective on the other tested cancer cell lines. The overall results provide valuable insights into using wireframe DNA origami as a highly stable possible carrier of Pt species for very long time-release purposes.

Spatial organization-dependent EphA2 transcriptional responses revealed by ligand nanocalipers.

Ligand binding induces extensive spatial reorganization and clustering of the EphA2 receptor at the cell membrane. It has previously been shown that the nanoscale spatial distribution of ligands modulates EphA2 receptor reorganization, activation and the invasive properties of cancer cells. However, intracellular signaling downstream of EphA2 receptor activation by nanoscale spatially distributed ligands has not been elucidated. Here, we used DNA origami nanostructures to control the positions of ephrin-A5 ligands at the nanoscale and investigated EphA2 activation and transcriptional responses following ligand binding. Using RNA-seq, we determined the transcriptional profiles of human glioblastoma cells treated with DNA nanocalipers presenting a single ephrin-A5 dimer or two dimers spaced 14, 40 or 100 nm apart. These cells displayed divergent transcriptional responses to the differing ephrin-A5 nano-organization. Specifically, ephrin-A5 dimers spaced 40 or 100 nm apart showed the highest levels of differential expressed genes compared to treatment with nanocalipers that do not present ephrin-A5. These findings show that the nanoscale organization of ephrin-A5 modulates transcriptional responses to EphA2 activation.

A computational framework for DNA sequencing microscopy.

We describe a method whereby microscale spatial information such as the relative positions of biomolecules on a surface can be transferred to a sequence-based format and reconstructed into images without conventional optics. Barcoded DNA "polymerase colony" (polony) amplification techniques enable one to distinguish specific locations of a surface by their sequence. Image formation is based on pairwise fusion of uniquely tagged and spatially adjacent polonies. The network of polonies connected by shared borders forms a graph whose topology can be reconstructed from pairs of barcodes fused during a polony cross-linking phase, the sequences of which are determined by recovery from the surface and next-generation (next-gen) sequencing. We developed a mathematical and computational framework for this principle called polony adjacency reconstruction for spatial inference and topology and show that Euclidean spatial data may be stored and transmitted in the form of graph topology. Images are formed by transferring molecular information from a surface of interest, which we demonstrated in silico by reconstructing images formed from stochastic transfer of hypothetical molecular markers. The theory developed here could serve as a basis for an automated, multiplexable, and potentially superresolution imaging method based purely on molecular information.

DNA rendering of polyhedral meshes at the nanoscale.

It was suggested more than thirty years ago that Watson-Crick base pairing might be used for the rational design of nanometre-scale structures from nucleic acids. Since then, and especially since the introduction of the origami technique, DNA nanotechnology has enabled increasingly more complex structures. But although general approaches for creating DNA origami polygonal meshes and design software are available, there are still important constraints arising from DNA geometry and sense/antisense pairing, necessitating some manual adjustment during the design process. Here we present a general method of folding arbitrary polygonal digital meshes in DNA that readily produces structures that would be very difficult to realize using previous approaches. The design process is highly automated, using a routeing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures. Moreover, unlike conventional origami designs built from close-packed helices, our structures have a more open conformation with one helix per edge and are therefore stable under the ionic conditions usually used in biological assays.

Interplay of Three G-Quadruplex Units in the KIT Promoter.

The proto-oncogene KIT encodes for a tyrosine kinase receptor, which is a clinically validated target for treating gastrointestinal stromal tumors. The KIT promoter contains a G-rich domain within a relatively long sequence potentially able to form three adjacent G-quadruplex (G4) units, namely, K2, SP, and K1. These G4 domains have been studied mainly as single quadruplex units derived from short truncated sequences and are currently considered promising targets for anticancer drugs, alternatively to the encoded protein. Nevertheless, the information reported so far does not contemplate the interplay between those neighboring G4s in the context of the whole promoter, possibly thwarting drug-discovery efforts. Here we report the structural and functional study of the KIT promoter core sequence, in both single- and double-stranded forms, which includes all three predicted G4 units. By preventing the formation of alternatively one or two G4 units and by combining biophysical techniques and biological assays, we show for the first time that these quadruplexes cannot be analyzed independently, but they are correlated to each other. Our data suggest that, while K2 and K1 G-rich sequences retain the ability to fold into parallel G4 motifs within a long sequence, the SP G-rich domain contributes to G4 structure only together with K2. Remarkably, we have found that, in the context of a dynamic equilibrium between the three G4 units, the G4 formed by K1 has the most significant influence on the structure stability and on the biological role of the whole promoter.

Solution-Controlled Conformational Switching of an Anchored Wireframe DNA Nanostructure.

Self-assembled DNA origami nanostructures have a high degree of programmable spatial control that enables nanoscale molecular manipulations. A surface-tethered, flexible DNA nanomesh is reported herein which spontaneously undergoes sharp, dynamic conformational transitions under physiological conditions. The transitions occur between two major macrostates: a spread state dominated by the interaction between the DNA nanomesh and the BSA/streptavidin surface and a surface-avoiding contracted state. Due to a slow rate of stochastic transition events on the order of tens of minutes, the dynamic conformations of individual structures can be detected in situ with DNA PAINT microscopy. Time series localization data with automated imaging processing to track the dynamically changing radial distribution of structural markers are combined. Conformational distributions of tethered structures in buffers with elevated pH exhibit a calcium-dependent domination of the spread state. This is likely due to electrostatic interactions between the structures and immobilized surface proteins (BSA and streptavidin). An interaction is observed in solution under similar buffer conditions with dynamic light scattering. Exchanging between solutions that promote one or the other state leads to in situ sample-wide transitions between the states. The technique herein can be a useful tool for dynamic control and observation of nanoscale interactions and spatial relationships.

Entirely enzymatic nanofabrication of DNA-protein conjugates.

While proteins are highly biochemically competent, DNA offers the ability to program, both reactions and the assembly of nanostructures, with a control that is unprecedented by any other molecule. Their joining: DNA-protein conjugates - offer the ability to combine the programmability of DNA with the competence of proteins to form novel tools enabling exquisite molecular control and the highest biological activity in one structure. However, in order for tools like these to become viable for biological applications, their production must be scalable, and an entirely enzymatic process is one way to achieve this. Here, we present a step in this direction: enzymatic production of DNA-protein conjugates using a new self-labeling tag derived from a truncated VirD2 protein of Agrobacterium tumefaciens. Using our previously reported MOSIC method for enzymatic ssDNA oligo production, we outline a pipeline for protein-DNA conjugates without the need for any synthetic chemistry in a one-pot reaction. Further, we validate HER2 staining using a completely enzymatically produced probe, enable the decoration of cell membranes and control of genetic expression. Establishing a method where protein-DNA conjugates can be made entirely using biological or enzymatic processing, opens a path to harvest these structures directly from bacteria and ultimately in-vivo assembly.

The interplay between national and parental unemployment in relation to adolescent life satisfaction in 27 countries: analyses of repeated cross-sectional school surveys.

BACKGROUND: Previous research shows that parental unemployment is associated with low life satisfaction in adolescents. It is unclear whether this translates to an association between national unemployment and adolescent life satisfaction, and whether such a contextual association is entirely explained by parental unemployment, or if it changes as a function thereof. For adults, associations have been shown between unemployment and mental health, including that national unemployment can affect mental health and life satisfaction of both the employed and the unemployed, but to different degrees. The aim of this paper is to analyse how national unemployment levels are related to adolescent life satisfaction, across countries as well as over time within a country, and to what extent and in what ways such an association depends on whether the individual's own parents are unemployed or not. METHODS: Repeated cross-sectional data on adolescents' (aged 11, 13 and 15 years, n = 386,402) life satisfaction and parental unemployment were collected in the Health Behaviour in School-aged Children (HBSC) survey, in 27 countries and 74 country-years, across 2001/02, 2005/06 and 2009/10 survey cycles. We linked this data to national harmonised unemployment rates provided by OECD and tested their associations using multilevel linear regression, including interaction terms between national and parental unemployment. RESULTS: Higher national unemployment rates were related to lower adolescent life satisfaction, cross-sectionally between countries but not over time within countries. The verified association was significant for adolescents with and without unemployed parents, but stronger so in adolescents with unemployed fathers or both parents unemployed. Having an unemployed father, mother och both parents was in itself related to lower life satisfaction. CONCLUSION: Living in a country with higher national unemployment seems to be related to lower adolescent life satisfaction, whether parents are unemployed or not, although stronger among adolescents where the father or both parents are unemployed. However, variation in unemployment over the years did not show an association with adolescent life satisfaction.

Spatial control of membrane receptor function using ligand nanocalipers.

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA.

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3'-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.

Computer-Aided Production of Scaffolded DNA Nanostructures from Flat Sheet Meshes.

The use of DNA as a nanoscale construction material has been a rapidly developing field since the 1980s, in particular since the introduction of scaffolded DNA origami in 2006. Although software is available for DNA origami design, the user is generally limited to architectures where finding the scaffold path through the object is trivial. Herein, we demonstrate the automated conversion of arbitrary two-dimensional sheets in the form of digital meshes into scaffolded DNA nanostructures. We investigate the properties of DNA meshes based on three different internal frameworks in standard folding buffer and physiological salt buffers. We then employ the triangulated internal framework and produce four 2D structures with complex outlines and internal features. We demonstrate that this highly automated technique is capable of producing complex DNA nanostructures that fold with high yield to their programmed configurations, covering around 70 % more surface area than classic origami flat sheets.

Self-assembly of DNA into nanoscale three-dimensional shapes.

Molecular self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase 'scaffold strand' that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometre scale.

An error correction strategy for image reconstruction by DNA sequencing microscopy.

By pairing adjacent molecules in situ and then mapping these pairs, DNA microscopy could substantially reduce the workload in spatial omics methods by directly inferring geometry from sequencing data alone. However, experimental artifacts can lead to errors in the adjacency data, which distort the spatial reconstruction. Here we describe a method to correct two such errors: spurious crosslinks formed between any two nodes, and fused nodes that are formed out of multiple molecules. We build on the principle that spatially close molecules should be connected and show that these errors violate this principle, allowing for their detection and correction. Our method corrects errors in simulated data, even in the presence of up to 20% errors, and proves to be more efficient at removing errors from experimental data than a read count filter. Integrating this method in DNA microscopy will substantially improve the accuracy of spatial reconstructions with lower data loss.

Effects of parental job loss on psychotropic drug use in children: Long-term effects, timing, and cumulative exposure.

Intra-family crossover effects triggered by job losses have received growing attention across scientific disciplines, but existing research has reached discrepant conclusions concerning if, and if so how, parental job losses affect child mental health. Drawing on sociological models of stress and life course epidemiology, we ask if parental job losses have long-term effects on child mental health, and if these effects are conditional on the timing of, or the cumulative exposure to, job losses. We use intergenerationally linked Swedish register data combined with entropy balance and structural nested mean models for the analyses. The data allow us to track 400,000 children over 14 years and thereby test different life-course models of cross-over effects. We identify involuntary job losses using information on workplace closures, thus reducing the risk of confounding. Results show that paternal but not maternal job loss significantly increases the risk of psychotropic drug use among children, that the average effects are modest in size (less than 4% in relative terms), that they may persist for up to five years, and that they are driven by children aged 6-10 years. Moreover, cumulative exposure to multiple job losses are more harmful than zero or one job loss.

Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force.

The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.