scholarly journals Therapeutic Nucleus-Access BNCT Drug Combined CD47-Targeting Gene Editing in Glioblastoma

2021 ◽  
Author(s):  
Jiejian Chen ◽  
Qi Dai ◽  
QiYao Yang ◽  
Xiaoyan Bao ◽  
Yi Zhou ◽  
...  
Keyword(s):  
Delivery System ◽  
Nuclear Translocation ◽  
Combined Treatment ◽  
Nuclear Targeting ◽  
Boron Neutron Capture ◽  
Checkpoint Pathway ◽  
Short Course ◽  
System Coupling ◽  
Insight Into ◽  
Clinical Drug

Abstract Glioblastoma is the most common brain primary malignant tumor with the highest mortality. Boron neutron capture therapy (BNCT) can efficiently kill cancer cells on the cellular scale, with high accuracy, short course and low side-effects, which is regarded as the most promising therapy for malignant brain tumors like glioma. As the keypoint of BNCT, all boron delivery agents currently in clinical use are beset by insufficient tumor uptake, especially in the tumor nucleus, which limits the clinical application of BNCT. In this study, nuclear targeting of boron is achieved by DOX-CB, consisting of doxorubicin (DOX) andcarborane (CB) utilizing the nuclear translocation property of DOX. The nucleus of GL261 cells takes up almost three times the concentration of boron required for BNCT. To further kill glioma and inhibit recurrence, a new multifunctional nanoliposome delivery system DOX-CB@lipo-pDNA-iRGDis constructed. It combines DOX-CB with immunotherapy strategy of blocking macrophage immune checkpoint pathway CD47-SIRPα by CRISPR-Cas9 system, coupling BNCT with immunotherapy simultaneously. Compared with clinical drug Borocaptate Sodium (BSH), DOX-CB@lipo-pDNA-iRGD significantly enhances the survival rate of tumor-bearing mice, reduces tumor stemness, and improves the prognosis. The excellent curative effect of this nanoliposome delivery system provides an insight into the combined treatment of BNCT.

scholarly journals The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

2016 ◽  
Vol 90 (13) ◽  
pp. 5860-5875 ◽  
Author(s):  
Eva Maria Borst ◽  
Rudolf Bauerfeind ◽  
Anne Binz ◽  
Thomas Min Stephan ◽  
Sebastian Neuber ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Fluorescent Protein ◽  
Unit Length ◽  
Start Codon ◽  
Nuclear Targeting ◽  
Reading Frame ◽  
A Genome ◽  
Genome Encapsidation ◽  
Insight Into

ABSTRACTSeveral essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

scholarly journals Translational Evaluation of a Dodecaborated Albumin Conjugate With Maleimide as a Boron Delivery System for a Neutron Capture Reaction to an F98 Rat Glioma Model

2021 ◽  
Author(s):  
Hideki Kashiwagi ◽  
Shinji Kawabata ◽  
Kohei Yoshimura ◽  
Yusuke Fukuo ◽  
Takuya Kanemitsu ◽  
...  
Keyword(s):  
Delivery System ◽  
Neutron Irradiation ◽  
Neutron Capture ◽  
Control Group ◽  
Clinical Settings ◽  
Neutron Capture Reaction ◽  
Capture Reaction ◽  
Rat Glioma ◽  
F98 Rat Glioma ◽  
Boron Neutron Capture

Abstract Boron neutron capture therapy (BNCT) is a biologically targeted, cell-selective particle irradiation therapy that utilizes the nuclear capture reaction of boron and neutron. Recently, accelerator neutron generators have been used in clinical settings, and expectations for developing new boron compounds are growing. In this study, we focused on serum albumin, a well-known drug delivery system, and developed maleimide-functionalized closo-dodecaborate albumin conjugate (MID-AC) as a boron carrying system for BNCT. Our biodistribution experiment involved F98 glioma-bearing rat brain tumor models systemically administered with MID-AC and demonstrated accumulation and long retention of boron. Our BNCT study with MID-AC observed statistically significant prolongation of the survival rate compared to the control groups, with results comparable to BNCT study with boronophenylalanine (BPA) which is the standard use of in clinical settings. Each median survival time was as follows: untreated control group; 24.5 days, neutron-irradiated control group; 24.5 days, neutron irradiation following 2.5 hours after termination of intravenous administration (i.v.) of BPA; 31.5 days, and neutron irradiation following 2.5 or 24 hours after termination of i.v. of MID-AC; 33.5 or 33.0 days, respectively. The biological effectiveness factor of MID-AC for F98 rat glioma was estimated based on these survival times and found to be higher to 12. This tendency was confirmed in BNCT 24 hours after MID-AC administration. MID-AC induces an efficient boron neutron capture reaction because the albumin contained in MID-AC is retained in the tumor and has a considerable potential to become an effective delivery system for BNCT in treating high-grade gliomas.

scholarly journals The role of phagocytosis-dependent activation of TFEB in the clearance of salmonella

2021 ◽  
Author(s):  
Erika Ospina Escobar
Keyword(s):  
Transcription Factor ◽  
Salmonella Typhimurium ◽  
Nuclear Translocation ◽  
Steady Increase ◽  
Phagosome Maturation ◽  
Salmonella Infection ◽  
Complex Interplay ◽  
Transcription Factor Eb ◽  
Insight Into

During phagocytosis, macrophages engulf and sequester pathogens into phagosomes. Phagosomes then fuse with acidic and degradative lysosomes to degrade the internalized pathogen. We previously demonstrated that phagocytosis of IgG-opsonized particles and non-opsonized E.coli causes activation of the Transcription Factor EB (TFEB), which enhances the expression of lysosomal genes, increases the degradative capacity of lysosomes and boosts bactericidal activity. However, pathogens like Salmonella typhimurium have evolved mechanisms to evade and/or alter phagosome maturation to promote their own survival. We investigated: i) whether pathogens like Salmonella can alter TFEB activation and ii) whether phagocytosis-dependent activation of TFEB can counteract the pathogenicity of microorganisms. Here, we show that non-viable (heat-killed) S. typhimurium, pathogenic (EHEC and UPEC) and non-pathogenic E.coli (DH5α) all caused TFEB nuclear translocation in RAW macrophages, while strikingly live S. typhimurium maintained TFEB in the cytosol in the first hours post-infection. By contrast, Salmonella mutants for ΔsifA, ΔsopD2, ΔphoP all triggered TFEB activation in the first hour of infection. However, Salmonella infection eventually triggered a steady increase in nuclear TFEB after 4 h of infection, suggesting a more complex interplay between TFEB and Salmonella infection. We dissected the importance of TFEB activation towards Salmonella survivability by pre-activating TFEB before infection within WT macrophages and macrophages with a CRISPR-based deletion of TFEB. Our work suggests that Salmonella actively interferes with TFEB signaling in order to enhance its own survival. These results could provide insight into using TFEB as a target for the clearance of infections.

scholarly journals Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections

2020 ◽  
Vol 14 (12) ◽  
pp. e0008930
Author(s):  
Andrea Schiefer ◽  
Marc P. Hübner ◽  
Anna Krome ◽  
Christine Lämmer ◽  
Alexandra Ehrens ◽  
...  
Keyword(s):  
Adult Worm ◽  
Neglected Tropical Diseases ◽  
Combined Treatment ◽  
Wuchereria Bancrofti ◽  
Drug Candidate ◽  
Treatment Regimens ◽  
Short Course ◽  
Filarial Nematodes ◽  
Product Profile

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal–adult-worm killing–treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4–5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.

scholarly journals Identification of the human c-myc protein nuclear translocation signal.

1988 ◽  
Vol 8 (10) ◽  
pp. 4048-4054 ◽  
Author(s):  
C V Dang ◽  
W M Lee
Keyword(s):  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Vero Cells ◽  
Embryo Cell ◽  
Nuclear Accumulation ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Chicken Muscle ◽  
Localization Signal ◽  
Muscle Pyruvate Kinase

We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.

scholarly journals REST/NRSF-Interacting LIM Domain Protein, a Putative Nuclear Translocation Receptor

2003 ◽  
Vol 23 (24) ◽  
pp. 9025-9031 ◽  
Author(s):  
Masahito Shimojo ◽  
Louis B. Hersh
Keyword(s):  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Protein A ◽  
Lim Domain ◽  
Nuclear Targeting ◽  
Lim Domains ◽  
Lim Domain Protein ◽  
Domain Protein ◽  
Nuclear Localization Sequences ◽  
Novel Protein

ABSTRACT The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors.

scholarly journals Cytoplasmic Localization of Wis1 MAPKK by Nuclear Export Signal Is Important for Nuclear Targeting of Spc1/Sty1 MAPK in Fission Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2651-2663 ◽  
Author(s):  
Aaron Ngocky Nguyen ◽  
Aminah D. Ikner ◽  
Mitsue Shiozaki ◽  
Sasha M. Warren ◽  
Kazuhiro Shiozaki
Keyword(s):  
Fission Yeast ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
Cytoplasmic Localization ◽  
Nuclear Targeting ◽  
Export Signal

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1 + does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.

scholarly journals Nuclear Targeting by the Growth Factor Midkine

2002 ◽  
Vol 22 (19) ◽  
pp. 6788-6796 ◽  
Author(s):  
Yoshihisa Shibata ◽  
Takashi Muramatsu ◽  
Makoto Hirai ◽  
Tatsuya Inui ◽  
Terutoshi Kimura ◽  
...  
Keyword(s):  
Growth Factor ◽  
Intracellular Signaling ◽  
Nuclear Translocation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Receptor Internalization ◽  
Heparin Binding ◽  
Nuclear Targeting ◽  
Density Lipoprotein Receptor ◽  
Localization Signal

ABSTRACT Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.

Sign in / Sign up

Export Citation Format

Share Document