In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems

2020 ◽  
Author(s):  
Wooli Bae ◽  
Guy-Bart V. Stan ◽  
Thomas E. Ouldridge
Keyword(s):  
Continuous Production ◽  
Autonomous Systems ◽  
Strand Displacement ◽  
Essential Components ◽  
Displacement Reactions ◽  
Feedback Control Systems ◽  
Molecular Computers ◽  
Rna Complexes

AbstractSynthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components – multi-stranded complexes known as “gates” – in situ, i.e. in living cells or other autonomous systems. Here, we propose the use of naturally occurring self-cleaving ribozymes to cut a single-stranded RNA transcript into a gate complex of shorter strands, thereby opening new possibilities for the autonomous and continuous production of RNA strands in a stoichiometrically and structurally controlled way.

In Vitro Human Tissues via Multi-material 3-D Bioprinting

2018 ◽  
Vol 46 (4) ◽  
pp. 209-215 ◽  
Author(s):  
David B. Kolesky ◽  
Kimberly A. Homan ◽  
Mark Skylar-Scott ◽  
Jennifer A. Lewis
Keyword(s):  
Kidney Tissue ◽  
Harvard University ◽  
Human Tissues ◽  
Osteogenic Lineage ◽  
Rapid Fabrication ◽  
Essential Components ◽  
Foundational Research

This paper highlights the foundational research on multi-material 3-D bioprinting of human tissues, for which the Lewis Bioprinting team at Harvard University was awarded the 2017 Lush Science Prize. The team's bioprinting platform enables the rapid fabrication of 3-D human tissues that contain all of the essential components found in their in vivo counterparts: cells, vasculature (or other tubular features) and extracellular matrix. The printed 3-D tissues are housed within a customised perfusion system and are subjected to controlled microphysiological environments over long durations (days to months). As exemplars, the team created a thick, stem cell-laden vascularised tissue that was controllably differentiated toward an osteogenic lineage in situ, and a 3-D kidney tissue that recapitulated the proximal tubule, a sub-unit of the nephron responsible for solute reabsorption. This highly versatile platform for manufacturing 3D human tissue in vitro opens new avenues for replacing animal models used to develop next-generation therapies, test toxicity and study disease pathology.

scholarly journals Configuring robust DNA strand displacement reactions for in situ molecular analyses

2011 ◽  
Vol 40 (7) ◽  
pp. 3289-3298 ◽  
Author(s):  
Dzifa Y. Duose ◽  
Ryan M. Schweller ◽  
Jan Zimak ◽  
Arthur R. Rogers ◽  
Walter N. Hittelman ◽  
...  
Keyword(s):  
Strand Displacement ◽  
Molecular Analyses ◽  
Dna Strand Displacement ◽  
Displacement Reactions ◽  
Dna Strand

scholarly journals Signal-Processing and Adaptive Prototissue Formation in Metabolic DNA Protocells

2021 ◽  
Author(s):  
Avik Samanta ◽  
Maximilian Hörner ◽  
Wei Liu ◽  
Wilfried Weber ◽  
Andreas Walther
Keyword(s):  
Signal Processing ◽  
Active Sites ◽  
Bond Cleavage ◽  
Soft Materials ◽  
Metabolic Reaction ◽  
Strand Displacement ◽  
Synthetic Cell ◽  
Displacement Reactions ◽  
Dna Strand

Abstract The fundamental life-defining processes in living cells, such as replication, division, adaptation, and tissue formation, take place via intertwined metabolic reaction networks orchestrating downstream signal processing in a confined, crowded environment with high precision. Hence, it is crucial to understand and reenact some of these functions in wholly synthetic cell-like entities (protocells) to envision designing soft-materials with life-like traits. Herein, we report on a programmable all-DNA protocell (PC) composed of a liquid DNA interior and a hydrogel-like shell, harboring DNAzyme active sites in the interior whose catalytic bond-cleaving activity leads to a downstream phenotype change in the protocells, as well as triggers prototissue formation. In this regard, we coupled several tools of DNA nanoscience, such as RNA cleavage, dynamic strand displacement reactions, and multivalent palindromic interactions, in a synchronize pathway so that the input signal can be processed inside the protocells and generate downstream cues giving rise to metabolic adaptive behavior. For example, the compartmentalized DNAzyme catalyzes the bond-cleavage of a substrate that releases a DNA strand in situ to trigger a strand displacement reaction at the shell of the protocells leading to a change in color resembling a “phenotype-like” change in cells, and finally to establish communication with other protocells via multivalent interactions.

Abstract 3437: Integrin-linked-kinase Binding Proteins Are Essential Components Of The Cardiac Mechanical Strech Sensor

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Andreas Hartkopf ◽  
Inken Huttner ◽  
Tillman Dahme ◽  
Benjamin Meder ◽  
Britta Vogel ◽  
...  
Keyword(s):  
Binding Proteins ◽  
In Situ Hybridisation ◽  
Cardiac Contractility ◽  
Down Regulation ◽  
Knock Down ◽  
Essential Components ◽  
Integrin Linked Kinase ◽  
Heart Contractility

The cardiac strech sensor enables the heart to adapt its force of contraction to continually changing demands. We recently identified in the zebrafish mutant main squeeze (msq) Integrin-Linked-Kinase (ILK) as a component of this strech sensor. Mutations in zebrafish ilk lead to down-regulation of stretch responsive cardiac genes and cause progressive heart failure. Antisense-mediated abrogation of zebrafish β-parvin, which forms complexes with ILK, phenocopies ilk mutant zebrafish. We here describe two novel ILK-binding-proteins, acting in concert with ILK to control cardiac contractility. In a yeast-to-hybrid-screen with a cardiac specific library we identified two ILK-binding proteins (ILK-bp-1 and ILK-bp-2). Just as we have recently shown for ILK, ILK-bp-1/2 are highly expressed in the zebrafish heart and localize at the sarcomeric Z-disc. To reveal the in vivo function of ILK-bp-1/2 we performed knock-down studies on zebrafish with antisense oligonucleotide injection. Similar to the phenotype of ILK-deficient zebrafish, the knock down of ILK-bp-1/2 leads to loss of cardiac contractility and, as shown by in-situ-hybridisation, to down-regulation of the stretch responsive gene anf. We analysed the ILK-bp-1/2 morphant hearts structurally and ultra-structurally. Just as we have recently shown for ilk deficient hearts, we found that their morphogenesis and myofibrillogenesis are normal, indicating that knock-down of ILK-binding-proteins does not induce a structural but rather a functional heart defect. Taken together, these results indicate, that next to β-parvin other ILK-binding proteins play important roles in the control of heart contractility. ILK and ILK-bp-1/2 act in concert to enable the heart to adapt itself to continually changing demands.

scholarly journals Signal-Processing and Adaptive Prototissue Formation in Metabolic DNA Protocells

2021 ◽  
Author(s):  
Avik Samanta ◽  
Maximilian Hörner ◽  
Wei Liu ◽  
Wilfried Weber ◽  
Andreas Walther
Keyword(s):  
Signal Processing ◽  
Active Sites ◽  
Bond Cleavage ◽  
Soft Materials ◽  
Metabolic Reaction ◽  
Strand Displacement ◽  
Synthetic Cell ◽  
Displacement Reactions ◽  
Dna Strand

The fundamental life-defining processes in living cells, such as replication, division, adaptation, and tissue formation, take place via intertwined metabolic reaction networks orchestrating downstream signal processing in a confined, crowded environment with high precision. Hence, it is crucial to understand and reenact some of these functions in wholly synthetic cell-like entities (protocells) to envision designing soft-materials with life-like traits. Herein, we report on a programmable all-DNA protocell (PC) composed of a liquid DNA interior and a hydrogel-like shell, harboring DNAzyme active sites in the interior whose catalytic bond-cleaving activity leads to a downstream phenotype change in the protocells, as well as triggers prototissue formation. In this regard, we coupled several tools of DNA nanoscience, such as RNA cleavage, dynamic strand displacement reactions, and multivalent palindromic interactions, in a synchronize pathway so that the input signal can be processed inside the protocells and generate downstream cues giving rise to metabolic adaptive behavior. For example, the compartmentalized DNAzyme catalyzes the bond-cleavage of a substrate that releases a DNA strand in situ to trigger a strand displacement reaction at the shell of the protocells leading to a change in color resembling a “phenotype-like” change in cells, and finally to establish communication with other protocells via multivalent interactions.

In vivo manipulation of dendritic cells to induce therapeutic immunity

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1676-1682 ◽  
Author(s):  
Miriam Merad ◽  
Tomoharu Sugie ◽  
Edgar G. Engleman ◽  
Lawrence Fong
Keyword(s):  
Dendritic Cells ◽  
Dendritic Cell ◽  
Antitumor Immunity ◽  
Flt3 Ligand ◽  
Essential Components ◽  
Tumor Protection ◽  
Immunostimulatory Dna

Efficient antigen presentation and T-cell priming are essential components of effective antitumor immunity. Dendritic cells are critical to both of these functions but to date no method has been devised that both targets antigen to these cells and activates them, in situ, in a manner that induces systemic immunity. In this study we combined a dendritic cell growth factor, Flt3 ligand, with a dendritic cell activator, immunostimulatory DNA, and a tumor antigen to activate and load dendritic cells in vivo. Initial studies showed that immunostimulatory DNA not only activates dendritic cells but also prolongs their survival in vivo and in vitro. Following treatment of mice with Flt3 ligand, coadministration of immunostimulatory DNA and antigen induced potent antitumor immunity, resulting in both tumor prevention and regression of existing tumors. CD8 cytotoxic T lymphocytes but not CD4 T cells were required for tumor protection. Natural killer cells also contributed to tumor protection. These results show that dendritic cells can be loaded with antigen and activated, in situ, and provide the basis for dendritic cell- targeted clinical strategies.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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