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Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signalling networks. Synthetic silicon-based circuitry similarly relies on spatial constraints to process information. Here, we show that spatial organization can be a similarly powerful design principle for overcoming limitations of speed and modularity in engineered molecular circuits. We create logic gates and signal transmission lines by spatially arranging reactive DNA hairpins on a DNA origami. Signal propagation is demonstrated across transmission lines of different lengths and orientations and logic gates are modularly combined into circuits that establish the universality of our approach. Because reactions preferentially occur between neighbours, identical DNA hairpins can be reused across circuits. Co-localization of circuit elements decreases computation time from hours to minutes compared to circuits with diffusible components. Detailed computational models enable predictive circuit design. We anticipate our approach will motivate using spatial constraints for future molecular control circuit designs. Fast and scalable molecular logic circuits can be created through the spatial organization of DNA hairpins on DNA origami scaffolds.

This article reviews recent progress in the development of cellular DNA nanotechnology, highlighting key potential applications such as DNA-based imaging probes, smart therapeutics, and drug delivery systems. The programmability of Watson–Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology — applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems — lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

The recent development of single-cell genomic techniques allows us to profile gene expression at the single-cell level easily, although many of these methods have limited throughput. Rosenberg et al. describe a strategy called split-pool ligation-based transcriptome sequencing, or SPLiT-seq, which uses combinatorial barcoding to profile single-cell transcriptomes without requiring the physical isolation of each cell. The authors used their method to profile >100,000 single-cell transcriptomes from mouse brains and spinal cords at 2 and 11 days after birth. Comparisons with in situ hybridization data on RNA expression from Allen Institute atlases linked these transcriptomes with spatial mapping, from which developmental lineages could be identified. Science , this issue p. 176 Single-cell analyses with SPLiT-seq (split-pool ligation-based transcriptome sequencing) elucidate development of the mouse nervous system. To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing, and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. More than 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation. Pseudotime analysis revealed transcriptional programs driving four developmental lineages, providing a snapshot of early postnatal development in the murine central nervous system. SPLiT-seq provides a path toward comprehensive single-cell transcriptomic analysis of other similarly complex multicellular systems.

Molecular machines that assemble polymers in a programmed sequence are fundamental to life. They are also an achievable goal of nanotechnology. Here, we report synthetic molecular machinery made from DNA that controls and records the formation of covalent bonds. We show that an autonomous cascade of DNA hybridization reactions can create oligomers, from building blocks linked by olefin or peptide bonds, with a sequence defined by a reconfigurable molecular program. The system can also be programmed to achieve combinatorial assembly. The sequence of assembly reactions and thus the structure of each oligomer synthesized is recorded in a DNA molecule, which enables this information to be recovered by PCR amplification followed by DNA sequencing. Molecular machines that assemble polymers in a programmed sequence are fundamental to life. Now, synthetic machinery built from DNA has been used to execute a molecular program that produces peptides, or olefin oligomers, with a defined sequence. The oligomeric product is linked to a double-stranded DNA product that records the sequence of reactions that were executed.

Objective: Experimental endotoxaemia induces subcutaneous adipose tissue inflammation and systemic insulin resistance in lean subjects. Glyceroneogenesis, by limiting free fatty acids (FFA) release from adipocytes, controls FFA homoeostasis and systemic insulin sensitivity. The roles of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in metabolic deregulation are intrinsically different. We compared the effect of lipopolysaccharide (LPS) on the inflammation profiles of SAT and VAT explants from lean women, as well as on glyceroneogenesis, to test whether these two fat depots have intrinsically different responses to this metabolic endotoxin. Design: Abdominal SAT and VAT explants from eight lean women were treated in vitro with LPS. Their inflammatory status was evaluated by cytokine gene expression and secretion; glyceroneogenesis was evaluated by cytosolic phosphoenolpyruvate carboxykinase activity and FFA vs glycerol release. Results: In the basal state, the cytokine status and expression of macrophage markers were lower in SAT than VAT. In the presence of 100 ng ml −1 LPS, SAT exhibited a strong inflammatory response (increased interleukin-6 and tumor necrosis factor-α expression) and increased release of FFA due to inhibition of glyceroneogenesis, whereas VAT was only mildly affected. The effects of LPS on both tissues were blocked by the nuclear factor-κB (NF-κB) inhibitor, parthenolide. A significant effect of LPS on VAT occurred only at 1 μg ml −1 LPS. Conclusion: SAT explants from lean women are more sensitive to LPS-induced NF-κB activation than are VAT explants, leading to a depot-specific dysfunction of FFA storage. As SAT is the major player in FFA homoeostasis, this SAT dysfunction could be associated with visceral fat hypertrophy and systemic lipid disorders.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Avian species are in rapid decline worldwide, with 1 in 8 species facing extinction, and in North America shorebird species are declining at a higher proportion than other groups. The nearshore ecosystem in Maine (USA) supports several bird species that rely on intertidal and subtidal zones, including high densities of migratory shorebirds and wintering waterfowl. Additionally, Acadia National Park (ANP) includes some of the country's highest densities of declining populations of Purple Sandpipers (Calidris maritima) and state threatened Harlequin Ducks (Histrionicus histrionicus). We investigated whether the distribution of candidate umbrella species overlapped with the distribution of 5 waterfowl taxa (Common Eiders [Somateria mollissima], Long-tailed Ducks [Clangula hyemalis], Black Scoters [Melanitta americana], Bucephala spp., and Mallards [Anas platyrhynchos]), thereby examining whether conservation actions (e.g., conservation closures) taken to preserve these 2 species of high conservation concern might also benefit the larger taxonomic groups to which they belong, a concept known as umbrella species conservation. To answer this question, bird abundance surveys were conducted within ANP during 2021 and 2022, and detection and occupancy were compared among the focal groups using a hypothesis-driven model. The occupancy distributions of our 5 waterfowl taxa revealed that sites with the highest occupancy estimates for Common Eiders, Long-tailed Ducks, and Black Scoters included sites occupied by Purple Sandpipers and Harlequin Ducks. Bucephala spp. and Mallard occupancy, however, only weakly overlapped with our candidate umbrella detections, occurring in sites with the lowest and mid-range occupancy estimates. Thus, any positive effect of conservation closures based on these 2 species of concern might impact some, but not all, species we surveyed. Including more survey locations occupied by Purple Sandpipers and Harlequin Ducks would be necessary to see if this pattern is generalizable across the region. Our low sample size for umbrella species detections, however, also highlights the limited utility of using rare species as umbrellas. Las especies de aves están en un declive rápido en todo el mundo, con 1 de cada 8 especies en riesgo de extinción y en Norteamérica las especies de aves costeras están declinando a una mayor proporción que otros grupos. El ecosistema litoral en Maine (EEUU) sostiene a varias especies de aves que dependen de zonas intermareal y submareales, incluyendo altas densidades de aves playeras migratorias y aves acuáticas invernales. Adicionalmente, Acadia National Park (ANP) incluye unas de las densidades más altas del país de las poblaciones en declive de correlimo Calidris maritima y de pato Histrionicus histrionicus, amenazados a nivel estatal. Estudiamos si la distribución de especies sombrilla candidatas sobrelapaba con la distribución de 5 taxa de aves acuáticas (eider Somateria mollissima, pato Clangula hyemalis, pato negro Melanitta americana, Bucephala spp. y pato Anas platyrhynchos), por tanto examinando si las acciones de conservación (como cierres de conservación) tomadas para preservar esas 2 especies en peligro crítico también podrían beneficiar a los grupos taxonómicos más amplios a los que pertenecen, un concepto conocido como conservación de especies sombrilla. Para responder a esta pregunta, se realizaron reconocimientos de abundancia de aves en ANP durante el 2021 y el 2022, y se comparó la detección y la ocupación entre grupos focales usando un modelo guiado por hipótesis. La distribución de ocupación de nuestras 5 taxa de aves acuáticas mostraron que los sitios con los mayores estimados de ocupación para eider S. mollissima, pato C. hyemalis y pato negro Melanitta americana incluían sitios ocupados por correlimo C. maritima y pato H. histrionicus. Sin embargo, la ocupación de Bucephala spp. y de pato A. platyrhynchos sólo tuvo un débil sobrelapamiento con la detección de nuestras candidatas a especies sombrilla, en sitios con estimados de ocupación de rango medio y bajo. Entonces, cualquier conclusión positiva del efecto de conservación basada en estas 2 especies de interés puede tener algún impacto sobre algunas, pero no todas, las especies del reconocimiento. Sería necesario incluir más localidades de reconocimiento ocupadas por correlimo Calidris maritima y pato H. histrionicus para ver si este patrón es generalizable en toda la región. Nuestro bajo número de reconocimientos para la detección de especies sombrilla, sin embargo, también recalca el uso limitado de usar especies raras como especies sombrilla. Palabras clave: correlimoCalidris maritima, cosecha de algas, ecosistemas costeros, ocupación de una sola especie, pato Histrionicus histrionicus.

Avian species are in rapid decline worldwide, with 1 in 8 species facing extinction, and in North America shorebird species are declining at a higher proportion than other groups. The nearshore ecosystem in Maine (USA) supports several bird species that rely on intertidal and subtidal zones, including high densities of migratory shorebirds and wintering waterfowl. Additionally, Acadia National Park (ANP) includes some of the country's highest densities of declining populations of Purple Sandpipers (Calidris maritima) and state threatened Harlequin Ducks (Histrionicus histrionicus). We investigated whether the distribution of candidate umbrella species overlapped with the distribution of 5 waterfowl taxa (Common Eiders [Somateria mollissima], Long-tailed Ducks [Clangula hyemalis], Black Scoters [Melanitta americana], Bucephala spp., and Mallards [Anas platyrhynchos]), thereby examining whether conservation actions (e.g., conservation closures) taken to preserve these 2 species of high conservation concern might also benefit the larger taxonomic groups to which they belong, a concept known as umbrella species conservation. To answer this question, bird abundance surveys were conducted within ANP during 2021 and 2022, and detection and occupancy were compared among the focal groups using a hypothesis-driven model. The occupancy distributions of our 5 waterfowl taxa revealed that sites with the highest occupancy estimates for Common Eiders, Long-tailed Ducks, and Black Scoters included sites occupied by Purple Sandpipers and Harlequin Ducks. Bucephala spp. and Mallard occupancy, however, only weakly overlapped with our candidate umbrella detections, occurring in sites with the lowest and mid-range occupancy estimates. Thus, any positive effect of conservation closures based on these 2 species of concern might impact some, but not all, species we surveyed. Including more survey locations occupied by Purple Sandpipers and Harlequin Ducks would be necessary to see if this pattern is generalizable across the region. Our low sample size for umbrella species detections, however, also highlights the limited utility of using rare species as umbrellas. Received 12 August 2023. Accepted 2 March 2024.