scholarly journals NERDSS: a non-equilibrium simulator for multibody self-assembly at the cellular scale

2019 ◽  
Author(s):  
Matthew J. Varga ◽  
Spencer Loggia ◽  
Yiben Fu ◽  
Osman N Yogurtcu ◽  
Margaret E. Johnson
Keyword(s):  
Self Assembly ◽  
Reaction Diffusion ◽  
High Yield ◽  
Sufficient Detail ◽  
Significant Barrier ◽  
Active Processes ◽  
Component Assembly ◽  
Non Equilibrium

AbstractCurrently, a significant barrier to building predictive models of cell-based self-assembly processes is that molecular models cannot capture minutes-long cellular dynamics that couple distinct components with active processes, while reaction-diffusion models lack sufficient detail for capturing assembly structures. Here we introduce the Non-Equilibrium Reaction-Diffusion Self-assembly Simulator (NERDSS), which addresses this gap by integrating a structure-resolved reaction-diffusion algorithm with rule-based model construction. By representing proteins as rigid, multi-site molecules that adopt well-defined orientations upon binding, NERDSS simulates formation of large reversible structures with sites that can be acted on by reaction rules. We show how NERDSS allows for directly comparing and optimizing models of multi-component assembly against time-dependent experimental data. Applying NERDSS to assembly steps in clathrin-mediated endocytosis, we capture how the formation of clathrin caged structures can be driven by modulating the strength of clathrin-clathrin interactions, by adding cooperativity, or by localizing clathrin to the membrane. NERDSS further predicts how clathrin lattice disassembly can be driven by enzymes that irreversibly change lipid populations on the membrane. By modeling viral lattice assembly and recapitulating oscillations in protein expression levels for a circadian clock model, we illustrate the wide usability and adaptability of NERDSS. NERDSS simulates user-defined assembly models that were previously inaccessible to existing software tools, with broad applications to predicting self-assembly in vivo and designing high-yield assemblies in vitro.

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.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals Development of In Situ Self-Assembly Nanoparticles to Encapsulate Lopinavir and Ritonavir for Long-Acting Subcutaneous Injection

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 904
Author(s):  
Irin Tanaudommongkon ◽  
Asama Tanaudommongkon ◽  
Xiaowei Dong
Keyword(s):  
Sustained Release ◽  
Trough Concentration ◽  
Self Assembly ◽  
Subcutaneous Injection ◽  
Drug Loading ◽  
Entrapment Efficiency ◽  
Long Acting

Most antiretroviral medications for human immunodeficiency virus treatment and prevention require high levels of patient adherence, such that medications need to be administered daily without missing doses. Here, a long-acting subcutaneous injection of lopinavir (LPV) in combination with ritonavir (RTV) using in situ self-assembly nanoparticles (ISNPs) was developed to potentially overcome adherence barriers. The ISNP approach can improve the pharmacokinetic profiles of the drugs. The ISNPs were characterized in terms of particle size, drug entrapment efficiency, drug loading, in vitro release study, and in vivo pharmacokinetic study. LPV/RTV ISNPs were 167.8 nm in size, with a polydispersity index of less than 0.35. The entrapment efficiency was over 98% for both LPV and RTV, with drug loadings of 25% LPV and 6.3% RTV. A slow release rate of LPV was observed at about 20% on day 5, followed by a sustained release beyond 14 days. RTV released faster than LPV in the first 5 days and slower than LPV thereafter. LPV trough concentration remained above 160 ng/mL and RTV trough concentration was above 50 ng/mL after 6 days with one subcutaneous injection. Overall, the ISNP-based LPV/RTV injection showed sustained release profiles in both in vitro and in vivo studies.

scholarly journals Force generation by protein–DNA co-condensation

2021 ◽  
Author(s):  
Thomas Quail ◽  
Stefan Golfier ◽  
Maria Elsner ◽  
Keisuke Ishihara ◽  
Vasanthanarayan Murugesan ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Self Assembly ◽  
Spider Silk ◽  
Regulatory Elements ◽  
Heterochromatin Formation ◽  
First Order Phase Transition ◽  
Dna Strand ◽  
First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

A Simple Silicon Based Nitric Oxide Sensor

1999 ◽  
Author(s):  
Marcelo Bariatto ◽  
Rogerio Furlan ◽  
Koiti Arakai ◽  
Jorge J. Santiago-Aviles
Keyword(s):  
Nitric Oxide ◽  
Amperometric Detection ◽  
Cellular Level ◽  
High Yield ◽  
Sensor Calibration ◽  
Nitric Oxide Sensor ◽  
Sensor Configuration

Abstract Nitric oxide (NO) is known to mediate many beneficial physiology processes, motivating its detection in vivo as well as in vitro. Electrochemical detection provides the required cellular level determination of NO among several other techniques. In this work, electrochemical micro-sensors for both types of detection, in vivo and in vitro, were developed, exploring the silicon planar technology, which presents high yield and reliability and also permits batch fabrication. The developed in vitro sensor features eight detection sites (10 μm × 10 μm microelectrodes), for determination of nitric oxide spatial distribution or multi-species analysis. Different electrochemical methods were applied to provide sensor calibration and chemical reproducibility. For in vivo analysis, the designed structures have a needle shape (40 μm thick) and they were silicon micro-machined by using plasma etching or etch stop techniques. Different configurations were designed and implemented, containing a number of detection microelectrodes that vary from 2 to 10. The amperometric detection of both nitric oxide and nitride (NO2−) — a molecule that causes an interference — were investigated by using the in vitro micro-sensor configuration. The need of a cationic exchanger (Nafion) was demonstrated in order to provide selectivity to NO for low concentrations. Also, the developed sensor has a sensitivity of 500 A/M.cm2 and a detection limit of 10 μM.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

scholarly journals Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

2021 ◽  
Vol 9 ◽  
Author(s):  
Ying Liu ◽  
Sabir Khan ◽  
Panpan Wu ◽  
Bowen Li ◽  
Lanlan Liu ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Target Genes ◽  
Antibacterial Activities ◽  
Saccharopolyspora Erythraea ◽  
High Yield ◽  
Yield Strain ◽  
Yield Improvement ◽  
Erythromycin Production

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

scholarly journals Charge influences substrate recognition and self-assembly of hydrophobic FG sequences

2016 ◽  
Author(s):  
Wesley G. Chen ◽  
Jacob Witten ◽  
Scott C. Grindy ◽  
Niels Holten-Andersen ◽  
Katharina Ribbeck
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Amino Acid Sequences ◽  
Nuclear Pore ◽  
Selective Binding ◽  
Charged Amino Acids ◽  
Essential Components ◽  
Transport Receptors

AbstractThe nuclear pore complex controls the passage of molecules via hydrophobic phenylalanine-glycine (FG) domains on nucleoporins. Such FG-domains consist of repeating units of FxFG, FG, or GLFG sequences, which can be interspersed with highly charged amino acid sequences. Despite the high density of charge exhibited in certain FG-domains, if and how charge influences FG-domain self-assembly and selective binding of nuclear transport receptors is largely unexplored. Studying how individual charged amino acids contribute to nuclear pore selectivity is challenging with modern in vivo and in vitro techniques due to the complexity of nucleoporin sequences. Here, we present a rationally designed approach to deconstruct essential components of nucleoporins down to 14 amino acid sequences. With these nucleoporin-based peptides, we systematically dissect how charge type and placement of charge influences self-assembly and selective binding of FG-containing gels. Specifically, we find that charge type determines which hydrophobic substrates FG sequences recognize while spatial localization of charge tunes hydrophobic self-assembly and receptor selectivity of FG sequences.

High Yield Expression of Recombinant Plasma Factors: Use of Recombinant Endoprotease Derivatives In Vivo and In Vitro

2006 ◽  
pp. 69-76
Author(s):  
U. Schlokat ◽  
A. Preininger ◽  
M. Himmelspach ◽  
G. Mohr ◽  
B. Fischer ◽  
...  
Keyword(s):  
High Yield ◽  
Plasma Factors

scholarly journals Polymorphonuclear Leukocytes Prepared by Continuous-Flow Filtration Leukapheresis: Viability and Function

Blood ◽  
1974 ◽  
Vol 44 (5) ◽  
pp. 707-713 ◽  
Author(s):  
Michael B. Harris ◽  
Isaac Djerassi ◽  
Elias Schwartz ◽  
Richard K. Root
Keyword(s):  
Continuous Flow ◽  
Polymorphonuclear Leukocytes ◽  
Cell Types ◽  
High Yield ◽  
Trypan Blue Exclusion ◽  
Flow Filtration ◽  
And Function ◽  
Combating Infection

Abstract Preparation of granulocytes for transfusion in high yield and relatively free of contamination by other cell types has been made possible by the technique of continuous-flow filtration leukapheresis (CFFL). Since previous work suggested that granulocytes collected in this manner may have impaired viability and function, a detailed study of the bactericidal, metabolic, and chemotactic properties of such cells was performed and compared to control cells obtained from the same donors prior to CFFL. The granulocyte percentage of the cell suspensions obtained by CFFL averaged 94.5% ± 1.5% compared to 82.5% ± 1.8% for the controls (p < 0.001) with viability of the PMNs determined by trypan blue exclusion being 97.5% ± 0.9% and 98.2% ± 0.5%, respectively. The phogocytic, metabolic (14C-I-glucose oxidation and protein iodination) and chemotactic properties of both cell types were equivalent in suspensions equalized for granulocyte content. These findings indicate that CFFL technique employed does not impair granulocyte viability or function in vitro. Studies of the in vivo survival and function of CFFL granulocytes are necessary to evaluate their efficacy in combating infection in severely leukopenic patients.

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