scholarly journals Light-activated communication in synthetic tissues

2016 ◽  
Vol 2 (4) ◽  
pp. e1600056 ◽  
Author(s):  
Michael J. Booth ◽  
Vanessa Restrepo Schild ◽  
Alexander D. Graham ◽  
Sam N. Olof ◽  
Hagan Bayley
Keyword(s):  
Lipid Bilayers ◽  
Three Dimensional ◽  
In Vitro Transcription ◽  
Translation System ◽  
System A ◽  
Synthetic Cells ◽  
3D Printed ◽  
Neuronal Transmission ◽  
Printed Patterns

We have previously used three-dimensional (3D) printing to prepare tissue-like materials in which picoliter aqueous compartments are separated by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional electrical communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way.

In Vitro Transcription/Translation System: A Versatile Tool in the Search for Missing Proteins

2015 ◽  
Vol 14 (9) ◽  
pp. 3441-3451 ◽  
Author(s):  
Péter Horvatovich ◽  
Ákos Végvári ◽  
Justin Saul ◽  
Jin G. Park ◽  
Ji Qiu ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Translation System ◽  
System A ◽  
Versatile Tool

scholarly journals Three-dimensionally printed polycaprolactone/multicomponent bioactive glass scaffolds for potential application in bone tissue engineering

2020 ◽  
Vol 6 (1) ◽  
pp. 57-69
Author(s):  
Amirhosein Fathi ◽  
Farzad Kermani ◽  
Aliasghar Behnamghader ◽  
Sara Banijamali ◽  
Masoud Mozafari ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
3D Printing ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Three Dimensional ◽  
Layer By Layer ◽  
Composite Scaffolds ◽  
3D Printed ◽  
Co Doped

AbstractOver the last years, three-dimensional (3D) printing has been successfully applied to produce suitable substitutes for treating bone defects. In this work, 3D printed composite scaffolds of polycaprolactone (PCL) and strontium (Sr)- and cobalt (Co)-doped multi-component melt-derived bioactive glasses (BGs) were prepared for bone tissue engineering strategies. For this purpose, 30% of as-prepared BG particles (size <38 μm) were incorporated into PCL, and then the obtained composite mix was introduced into a 3D printing machine to fabricate layer-by-layer porous structures with the size of 12 × 12 × 2 mm3.The scaffolds were fully characterized through a series of physico-chemical and biological assays. Adding the BGs to PCL led to an improvement in the compressive strength of the fabricated scaffolds and increased their hydrophilicity. Furthermore, the PCL/BG scaffolds showed apatite-forming ability (i.e., bioactivity behavior) after being immersed in simulated body fluid (SBF). The in vitro cellular examinations revealed the cytocompatibility of the scaffolds and confirmed them as suitable substrates for the adhesion and proliferation of MG-63 osteosarcoma cells. In conclusion, 3D printed composite scaffolds made of PCL and Sr- and Co-doped BGs might be potentially-beneficial bone replacements, and the achieved results motivate further research on these materials.

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

Algorithm to Reduce Leading and Lagging in Conformal Direct-Print

2018 ◽  
Vol 140 (10) ◽  
Author(s):  
Morteza Vatani ◽  
Faez Alkadi ◽  
Jae-Won Choi
Keyword(s):  
Additive Manufacturing ◽  
3D Printing ◽  
3D Model ◽  
Three Dimensional ◽  
Motion Direction ◽  
B Spline ◽  
And Topology ◽  
Printing System ◽  
3D Printed ◽  
Printed Patterns

A novel additive manufacturing algorithm was developed to increase the consistency of three-dimensional (3D) printed curvilinear or conformal patterns on freeform surfaces. The algorithm dynamically and locally compensates the nozzle location with respect to the pattern geometry, motion direction, and topology of the substrate to minimize lagging or leading during conformal printing. The printing algorithm was implemented in an existing 3D printing system that consists of an extrusion-based dispensing module and an XYZ-stage. A dispensing head is fixed on a Z-axis and moves vertically, while the substrate is installed on an XY-stage and moves in the x–y plane. The printing algorithm approximates the printed pattern using nonuniform rational B-spline (NURBS) curves translated directly from a 3D model. Results showed that the proposed printing algorithm increases the consistency in the width of the printed patterns. It is envisioned that the proposed algorithm can facilitate nonplanar 3D printing using common and commercially available Cartesian-type 3D printing systems.

scholarly journals Evaluation of the 3D Printing Accuracy of a Dental Model According to Its Internal Structure and Cross-Arch Plate Design: An In Vitro Study

Materials ◽  
2020 ◽  
Vol 13 (23) ◽  
pp. 5433
Author(s):  
Seung-Ho Shin ◽  
Jung-Hwa Lim ◽  
You-Jung Kang ◽  
Jee-Hwan Kim ◽  
June-Sung Shim ◽  
...  
Keyword(s):  
3D Printing ◽  
Internal Structure ◽  
Three Dimensional ◽  
In Vitro Study ◽  
Thick Shell ◽  
Plate Design ◽  
Dental Model ◽  
3D Printed ◽  
Modeling Data

The amount of photopolymer material consumed during the three-dimensional (3D) printing of a dental model varies with the volume and internal structure of the modeling data. This study analyzed how the internal structure and the presence of a cross-arch plate influence the accuracy of a 3D printed dental model. The model was designed with a U-shaped arch and the palate removed (Group U) or a cross-arch plate attached to the palate area (Group P), and the internal structure was divided into five types. The trueness and precision were analyzed for accuracy comparisons of the 3D printed models. Two-way ANOVA of the trueness revealed that the accuracy was 135.2 ± 26.3 µm (mean ± SD) in Group U and 85.6 ± 13.1 µm in Group P. Regarding the internal structure, the accuracy was 143.1 ± 46.8 µm in the 1.5 mm-thick shell group, which improved to 111.1 ± 31.9 µm and 106.7 ± 26.3 µm in the roughly filled and fully filled models, respectively. The precision was 70.3 ± 19.1 µm in Group U and 65.0 ± 8.8 µm in Group P. The results of this study suggest that a cross-arch plate is necessary for the accurate production of a model using 3D printing regardless of its internal structure. In Group U, the error during the printing process was higher for the hollowed models.

scholarly journals Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

mAbs ◽  
2012 ◽  
Vol 4 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Gang Yin ◽  
Eudean D. Garces ◽  
Junhao Yang ◽  
Juan Zhang ◽  
Cuong Tran ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Antibody Fragments ◽  
Translation System

scholarly journals Identification of a talin binding site in the cytoskeletal protein vinculin.

1989 ◽  
Vol 109 (6) ◽  
pp. 2917-2927 ◽  
Author(s):  
P Jones ◽  
P Jackson ◽  
G J Price ◽  
B Patel ◽  
V Ohanion ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Cytoskeletal Protein ◽  
Translation System ◽  
Cos Cells ◽  
Cell Matrix ◽  
Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

scholarly journals Vaccinia Virus F1L Protein Is a Tail-Anchored Protein That Functions at the Mitochondria To Inhibit Apoptosis

2005 ◽  
Vol 79 (2) ◽  
pp. 1084-1098 ◽  
Author(s):  
Tara L. Stewart ◽  
Shawn T. Wasilenko ◽  
Michele Barry
Keyword(s):  
Amino Acid ◽  
Vaccinia Virus ◽  
Transmembrane Domain ◽  
In Vitro Transcription ◽  
Translation System ◽  
Reading Frame ◽  
Protease Digestion ◽  
Necessary And Sufficient ◽  
Virus Survival

ABSTRACT Members of the poxvirus family encode multiple immune evasion proteins, including proteins that regulate apoptosis. We recently identified one such protein, F1L, encoded by vaccinia virus, the prototypic member of the poxvirus family. F1L localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome c, the pivotal commitment step in the apoptotic cascade. Sequence analysis of the F1L open reading frame revealed a C-terminal motif composed of a 12-amino-acid transmembrane domain flanked by positively charged lysines, followed by an 8-amino-acid hydrophilic tail. By generating a series of F1L deletion constructs, we show that the C-terminal domain is necessary and sufficient for localization of F1L to the mitochondria. In addition, mutation of lysines 219 and 222 downstream of the C-terminal transmembrane domain resulted in altered localization of F1L to the endoplasmic reticulum. Using F1L protein generated in an in vitro transcription-translation system, we found that F1L was posttranslationally inserted into mitochondria and tightly associated with mitochondrial membranes as demonstrated by resistance to alkaline extraction. Sensitivity to protease digestion showed that the N terminus of F1L was exposed to the cytoplasm. Utilizing various F1L deletion constructs, we found that F1L localization to the mitochondria was necessary to inhibit apoptosis, since constructs that no longer localized to the mitochondria had reduced antiapoptotic ability. Our studies show that F1L is a new member of the tail-anchored protein family that localizes to mitochondria during virus infection and inhibits apoptosis as a means to enhance virus survival.

scholarly journals Role of YidC in folding of polytopic membrane proteins

2004 ◽  
Vol 165 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Shushi Nagamori ◽  
Irina N. Smirnova ◽  
H. Ronald Kaback
Keyword(s):  
Membrane Proteins ◽  
Three Dimensional ◽  
In Vitro Transcription ◽  
Dimensional Structure ◽  
Lipid Phase ◽  
Lactose Permease ◽  
Primary Role ◽  
Protein Insertion

YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding. Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY. By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes. Moreover, LacY also folds improperly in proteoliposomes prepared without YidC. However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly. The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

scholarly journals Geometry sensing by self-organized protein patterns

2012 ◽  
Vol 109 (38) ◽  
pp. 15283-15288 ◽  
Author(s):  
Jakob Schweizer ◽  
Martin Loose ◽  
Mike Bonny ◽  
Karsten Kruse ◽  
Ingolf Mönch ◽  
...  
Keyword(s):  
Systems Approach ◽  
Protein Pattern ◽  
Three Dimensional ◽  
Reaction Diffusion ◽  
Self Organization ◽  
Protein Patterns ◽  
Intracellular Space ◽  
System A ◽  
Experimental Findings

In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction–diffusion system.

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