scholarly journals Phage Display of Adenovirus Type 5 Fiber Knob as a Tool for Specific Ligand Selection and Validation

2001 ◽  
Vol 75 (15) ◽  
pp. 7107-7113 ◽  
Author(s):  
Alexander Pereboev ◽  
Larisa Pereboeva ◽  
David T. Curiel
Keyword(s):  
Conformational Changes ◽  
High Efficiency ◽  
A Priori ◽  
Adenovirus Type ◽  
Target Cells ◽  
Genetic Modifications ◽  
Filamentous Bacteriophages ◽  
Serotype 5

ABSTRACT Adenovirus (Ad) vectors are most potent for use as gene delivery vehicles to infect human cells in vitro and in vivo with high efficiency. The main limitation in utilization of Ad as a gene transfer vector is the lack of specificity. Genetic modifications of Ad capsid proteins resulting in incorporation of foreign polypeptide ligand sequences can redirect the vector towards target cells. However, in many cases the incorporated ligands lose specificity or lead to conformational changes influencing virion integrity. In order to select target-specific ligands a priori structurally compatible with Ad, we propose a system for displaying polypeptide sequences in the context of the Ad fiber knob on the surfaces of filamentous bacteriophages. To establish this concept, we displayed the wild-type Ad serotype 5 knob and knobs containing c-Myc epitopes and six-histidine sequences in the pJuFo phage system. The knobs remained trimeric and bound the coxsackievirus-Ad receptor, and the phage knob-displayed ligands recognized and bound their cognates in the phage-displayed knob context. Further development of this system may be useful for candidate ligand fidelity and Ad structural compatibility validation prior to Ad modification.

scholarly journals Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2656-2664 ◽  
Author(s):  
Raul Alba ◽  
Angela C. Bradshaw ◽  
Lynda Coughlan ◽  
Laura Denby ◽  
Robert A. McDonald ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Adenovirus Type ◽  
Factor X ◽  
High Affinity ◽  
Serotype 5 ◽  
Adenoviral Transduction ◽  
High Doses ◽  
Immunohistochemical Studies

AbstractA major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c+, ER-TR7+, and MAdCAM-1+ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46+ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

scholarly journals Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo

2017 ◽  
Vol 312 (1) ◽  
pp. L110-L121 ◽  
Author(s):  
Duo Zhang ◽  
Heedoo Lee ◽  
Ziwen Zhu ◽  
Jasleen K. Minhas ◽  
Yang Jin
Keyword(s):  
High Efficiency ◽  
Therapeutic Interventions ◽  
Target Cells ◽  
Transfection Method ◽  
Exosomal Mirnas ◽  
Mother Cells ◽  
Antisense Oligos ◽  
Exosomal Mirna

Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals Combined In Vitro and In Vivo Approaches to Propose a Putative Adverse Outcome Pathway for Acute Lung Inflammation Induced by Nanoparticles: A Study on Carbon Dots

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 180
Author(s):  
Maud Weiss ◽  
Jiahui Fan ◽  
Mickaël Claudel ◽  
Luc Lebeau ◽  
Françoise Pons ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Lung Inflammation ◽  
Adverse Outcome ◽  
Cellular Responses ◽  
Target Cells ◽  
Inflammasome Activation ◽  
Adverse Outcome Pathway ◽  
Acute Lung Inflammation

With the growth of nanotechnologies, concerns raised regarding the potential adverse effects of nanoparticles (NPs), especially on the respiratory tract. Adverse outcome pathways (AOP) have become recently the subject of intensive studies in order to get a better understanding of the mechanisms of NP toxicity, and hence hopefully predict the health risks associated with NP exposure. Herein, we propose a putative AOP for the lung toxicity of NPs using emerging nanomaterials called carbon dots (CDs), and in vivo and in vitro experimental approaches. We first investigated the effect of a single administration of CDs on mouse airways. We showed that CDs induce an acute lung inflammation and identified airway macrophages as target cells of CDs. Then, we studied the cellular responses induced by CDs in an in vitro model of macrophages. We observed that CDs are internalized by these cells (molecular initial event) and induce a series of key events, including loss of lysosomal integrity and mitochondrial disruption (organelle responses), as well as oxidative stress, inflammasome activation, inflammatory cytokine upregulation and macrophage death (cellular responses). All these effects triggering lung inflammation as tissular response may lead to acute lung injury.

scholarly journals A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

scholarly journals Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

2001 ◽  
Vol 75 (21) ◽  
pp. 10054-10064 ◽  
Author(s):  
Jerg Schmidt ◽  
Volker Gerdts ◽  
Jörg Beyer ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Host Range ◽  
Pseudorabies Virus ◽  
Natural Host ◽  
Target Cells ◽  
Glycoprotein D ◽  
Wild Type ◽  
Cellular Receptors ◽  
Receptor Usage

ABSTRACT Infection of cells by herpesviruses is initiated by the interaction of viral envelope glycoproteins with cellular receptors. In the alphaherpesvirus pseudorabies virus (PrV), the causative agent of Aujeszky's disease in pigs, the essential glycoprotein D (gD) mediates secondary attachment of virions to target cells by binding to newly identified cellular receptors (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618–1620, 1998). However, in the presence of compensatory mutations, infection can also occur in the absence of gD, as evidenced by the isolation in cell culture of an infectious gD-negative PrV mutant (PrV-gD− Pass) (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). PrV-gD− Pass is replication competent with an only moderate reduction in specific infectivity but appears to bind to receptors different from those recognized by wild-type PrV (A. Karger, J. Schmidt, and T. C. Mettenleiter, J. Virol. 72:7341–7348, 1998). To analyze whether this alteration in receptor usage in vitro influences infection in vivo, the model host mouse and the natural host pig were intranasally infected with PrV-gD− Pass and were compared to animals infected by wild-type PrV. For mice, a comparable progress of disease was observed, and all animals infected with mutant virus died, although they exhibited a slight delay in the onset of symptoms and, correspondingly, a longer time to death. In contrast, whereas wild-type PrV-infected pigs showed clinical signs and histological and histopathological findings typical of PrV infection, no signs of disease were observed after infection with PrV-gD− Pass. Moreover, in these animals, virus-infected cells were not detectable by immunohistochemical staining of different organ samples and no virus could be isolated from nasal swabs. Mutations in glycoproteins B and H were found to correlate with, and probably contribute to, gD-independent infectivity. In conclusion, although PrV-gD− Pass is virulent in mice, it is apparently unable to infect the natural host, the pig. This altered host range in vivo correlates with a difference of receptor usage in vitro and demonstrates for the first time the importance of gD receptors in alphaherpesvirus infection of an animal host.

Sign in / Sign up

Export Citation Format

Share Document