A Rapid Self-Assembly Peptide Hydrogel for Recruitment and Activation of Immune Cells

Self-assembly peptide nanotechnology has attracted much attention due to its regular and orderly structure and diverse functions. Most of the existing self-assembly peptides can form aggregates with specific structures only under specific conditions and their assembly time is relatively long. They have good biocompatibility but no immunogenicity. To optimize it, a self-assembly peptide named DRF3 was designed. It contains a hydrophilic and hydrophobic surface, using two N-terminal arginines, leucine, and two c-terminal aspartate and glutamic acid. Meanwhile, the c-terminal of the peptide was amidated, so that peptide segments were interconnected to increase diversity. Its characterization, biocompatibility, controlled release effect on antigen, immune cell recruitment ability, and antitumor properties were examined here. Congo red/aniline blue staining revealed that peptide hydrogel DRF3 could be immediately gelled in PBS. The stable β-sheet secondary structure of DRF3 was confirmed by circular dichroism spectrum and IR spectra. The observation results of cryo-scanning electron microscopy, transmission electron microscopy, and atomic force microscopy demonstrated that DRF3 formed nanotubule-like and vesicular structures in PBS, and these structures interlaced with each other to form ordered three-dimensional nanofiber structures. Meanwhile, DRF3 showed excellent biocompatibility, could sustainably and slowly release antigens, recruit dendritic cells and promote the maturation of dendritic cells (DCs) in vitro. In addition, DRF3 has a strong inhibitory effect on clear renal cell carcinoma (786-0). These results provide a reliable basis for the application of peptide hydrogels in biomedical and preclinical trials.

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Lymph node follicle formation and vaccination responses reconstituted in vitro in a human Organ Chip

10.1101/806505 ◽

2019 ◽

Cited By ~ 1

Author(s):

Girija Goyal ◽

Bruce Bausk ◽

Pranav Prabhala ◽

Liangxia Xie ◽

Danielle Curran ◽

...

Keyword(s):

Lymph Node ◽

Plasma Cell ◽

Self Assembly ◽

Immune Cell ◽

Cytidine Deaminase ◽

Cell Formation ◽

Three Dimensional ◽

Human Organ ◽

Vaccine Responses

ABSTRACTCandidate vaccines and immunotherapeutic drugs often fail in clinical trials as human lymph node (LN) physiology is not faithfully modeled in animal models or immune cell cultures. Here we describe a microfluidic Organ Chip culture device that supports self-assembly of human blood-derived B and T lymphocytes into three-dimensional (3D), germinal center-like lymphoid follicles (LFs) containing Activation-Induced Cytidine Deaminase (AID) expressing lymphocytes. These microengineered LFs support plasma cell differentiation upon activation with IL-4 and CD40 agonistic antibody (AB) or inactivated S. aureus Cowan I (SAC). Immunization of the human LN chip with a quadrivalent split virion influenza vaccine resulted in plasma cell formation, viral strain-specific anti-hemagglutinin immunoglobulin G (IgG) production, and a secreted cytokine profile that recapitulates serum responses of vaccinated humans. Thus, the human LN chip may provide a new tool to study human immune reactions, evaluate vaccine responses, and validate the efficacies and toxicities of immunotherapies in vitro.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

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.

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An Analysis Based on Molecular Docking and Molecular Dynamics Simulation Study of Bromelain as Anti-SARS-CoV-2 Variants

Frontiers in Pharmacology ◽

10.3389/fphar.2021.717757 ◽

2021 ◽

Vol 12 ◽

Cited By ~ 1

Author(s):

Trina Ekawati Tallei ◽

Fatimawali ◽

Afriza Yelnetty ◽

Rinaldi Idroes ◽

Diah Kusumawaty ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Docking ◽

Molecular Dynamics Simulation ◽

Three Dimensional ◽

Md Simulations ◽

Inhibitory Effect ◽

Dynamics Simulation ◽

Novel Coronavirus

The rapid spread of a novel coronavirus known as SARS-CoV-2 has compelled the entire world to seek ways to weaken this virus, prevent its spread and also eliminate it. However, no drug has been approved to treat COVID-19. Furthermore, the receptor-binding domain (RBD) on this viral spike protein, as well as several other important parts of this virus, have recently undergone mutations, resulting in new virus variants. While no treatment is currently available, a naturally derived molecule with known antiviral properties could be used as a potential treatment. Bromelain is an enzyme found in the fruit and stem of pineapples. This substance has been shown to have a broad antiviral activity. In this article, we analyse the ability of bromelain to counteract various variants of the SARS-CoV-2 by targeting bromelain binding on the side of this viral interaction with human angiotensin-converting enzyme 2 (hACE2) using molecular docking and molecular dynamics simulation approaches. We have succeeded in making three-dimensional configurations of various RBD variants using protein modelling. Bromelain exhibited good binding affinity toward various variants of RBDs and binds right at the binding site between RBDs and hACE2. This result is also presented in the modelling between Bromelain, RBD, and hACE2. The molecular dynamics (MD) simulations study revealed significant stability of the bromelain and RBD proteins separately up to 100 ns with an RMSD value of 2 Å. Furthermore, despite increases in RMSD and changes in Rog values of complexes, which are likely due to some destabilized interactions between bromelain and RBD proteins, two proteins in each complex remained bonded, and the site where the two proteins bind remained unchanged. This finding indicated that bromelain could have an inhibitory effect on different SARS-CoV-2 variants, paving the way for a new SARS-CoV-2 inhibitor drug. However, more in vitro and in vivo research on this potential mechanism of action is required.

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Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy

Frontiers in Chemistry ◽

10.3389/fchem.2020.628433 ◽

2021 ◽

Vol 8 ◽

Author(s):

Floris J. van Dalen ◽

Thomas Bakkum ◽

Tyrza van Leeuwen ◽

Mirjam Groenewold ◽

Edgar Deu ◽

...

Keyword(s):

Electron Microscopy ◽

Dendritic Cells ◽

Immune Cell ◽

Ultrastructural Level ◽

Cross Reactivity ◽

Protein Profiling ◽

Cathepsin S ◽

Probe Design ◽

Competitive Activity ◽

Lysosomal Cysteine Protease

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.

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Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

The Journal of Cell Biology ◽

10.1083/jcb.202009146 ◽

2021 ◽

Vol 220 (3) ◽

Cited By ~ 1

Author(s):

Michal Wieczorek ◽

Shih-Chieh Ti ◽

Linas Urnavicius ◽

Kelly R. Molloy ◽

Amol Aher ◽

...

Keyword(s):

Electron Microscopy ◽

Self Assembly ◽

The Self ◽

Dependent Manner ◽

Guanine Nucleotide ◽

Partial Cone ◽

Ring Complex ◽

Microtubule Networks ◽

And Function

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.

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Inhibitory effect of resveratrol on the growth and angiogenesis of human endometrial tissue in an In Vitro three-dimensional model of endometriosis

Reproductive Biology ◽

10.1016/j.repbio.2020.07.012 ◽

2020 ◽

Vol 20 (4) ◽

pp. 484-490

Author(s):

Mohammad Rasool Khazaei ◽

Zahra Rashidi ◽

Farzaneh Chobsaz ◽

Elham Niromand ◽

Mozafar Khazaei

Keyword(s):

Three Dimensional ◽

Inhibitory Effect ◽

Endometrial Tissue ◽

Dimensional Model ◽

Three Dimensional Model

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Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma.

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1710 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1710-1725 ◽

Cited By ~ 43

Author(s):

S Tsukita ◽

T Kobayashi ◽

G Matsumoto

Keyword(s):

Electron Microscopy ◽

Three Dimensional ◽

Preceding Paper ◽

Microscopic Analysis ◽

Giant Axon ◽

Squid Giant Axon ◽

Molecular Organization ◽

Fixation Method ◽

Morphological Identification

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.

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Chapter 17 Electron Microscopy of Collagen Fibril Structure In Vitro and In Vivo Including Three-Dimensional Reconstruction

Methods in Cell Biology - Introduction to Electron Microscopy for Biologists ◽

10.1016/s0091-679x(08)00417-2 ◽

2008 ◽

pp. 319-345 ◽

Cited By ~ 29

Author(s):

Tobias Starborg ◽

Yinhui Lu ◽

Karl E. Kadler ◽

David F. Holmes

Keyword(s):

Electron Microscopy ◽

Collagen Fibril ◽

Three Dimensional ◽

Three Dimensional Reconstruction ◽

Fibril Structure ◽

Dimensional Reconstruction

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Synthesis of Thermo-Responsive Block-Graft Copolymer Based on PCL and PEG Analogs, and Preparation of Hydrogel via Click Chemistry

Polymers ◽

10.3390/polym11050765 ◽

2019 ◽

Vol 11 (5) ◽

pp. 765 ◽

Cited By ~ 1

Author(s):

Pei Shang ◽

Jie Wu ◽

Xiaoyu Shi ◽

Zhidan Wang ◽

Fei Song ◽

...

Keyword(s):

Electron Microscopy ◽

Graft Copolymer ◽

Self Assembly ◽

Three Dimensional ◽

Cycloaddition Reaction ◽

Temperature Response ◽

Gel Permeation Chromatography ◽

Proton Nuclear Magnetic Resonance ◽

Fluorescence Probes ◽

Different Temperatures

Thermo-responsive cross-linkable mPEG-b-[PCL-g-(MEO2MA-co-OEGMA)]-b-mPEG was synthesized by ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). Then, the cross-linkable block-graft copolymer was used to prepare hydrogel via a copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction. The chemical structure and composition of copolymer were characterized by proton nuclear magnetic resonance (1H NMR), Fourier-transform infrared (FT-IR) and gel permeation chromatography (GPC). The self-assembly behaviors of the copolymer in aqueous solution were studied by UV spectrophotometer, fluorescence probes, the surface tension method, dynamic light scattering, and transmission electron microscopy. The results proved that the copolymer has excellent solubility and better temperature response. The three-dimensional network structure of the gels, observed by scanning electron microscopy at different temperatures, indicated that the gels have temperature response.

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