Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro- Inflammation in Brain, Cognitive Deficits,and Alterations in Apurinic Endonuclease 1

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphical abstract

We used O-GNR-PEG-DSPE as a reliable, non-toxic vehicle for delivery of APE-1 inhibiting Lucanthone into GBM tumor cell lines. LUC-O-GNR-PEG-DSPE particles showed 60% or more uptake by CMV/U251 and A1-5/CMV/U251 where as the uptake by MCF7 and normal CG4 glial cells was much lower (38% and 29% respectively). Different concentrations of Luc (5–80 μM) loaded onto O-GNR-PEG-DSPE showed lower toxicity in the exposed cells compared to the free drug, due to possible slow release of the drug from this particle, which ensures minimum non-specific release of the drug from the particle once it is injected in vivo.
http://ars.els-cdn.com/content/image/1-s2.0-S1549963414004249-fx1.jpg

Elafin prevents lipopolysaccharide-induced AP-1 and NF-kB activation via an effect on the ubiquitin-proteasome pathway

In this paper we follow on from our research into SLPI by assessing the immunomodulatory activity of elafin - an antiprotease related to SLPI and also present on the respiratory tract. We demonstrate for the first time that exogenously applied elafin inhibits lipopolysaccharide-induced activation of the NF-kappaB and AP-1 pathways in monocytes. I designed this project and supervised Marcus Butler during his MD thesis.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA, Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA, As well as strand breaks, these sites may contain multiple damaged bases, We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH radicals with either Co-60 gamma-rays or alpha-particles from a Pu-238 source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1x10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp, This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute similar to 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

Fasciola hepatica:Histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks

Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1-4. days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the testis of untreated Cullompton (TCBZ-S) and Sligo Type 2 (TCBZ-R) flukes, which exhibit abnormal spermatogenesis and spermiogenesis, respectively. This was attributed to apoptosis and to heterophagy of effete germ line cells by the sustentacular tissue. It is concluded that demonstration of apoptosis by in situ hybridisation using the TUNEL method on sections of 1-4. days in vivo TCBZ-treated F. hepatica can contribute to the diagnosis of TCBZ resistance in field outbreaks of fasciolosis. © 2012 Elsevier B.V.

The Arabidopsis microtubule-associated protein AtMAP65-1: Molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.

Immunological homologues of the Arabidopsis thaliana beta 1 tubulin are polyglutamylated in Nicotiana tabacum

Peptide-specific antibody AABI, raised to the C-terminal 13 amino acids of Arabidopsis thaliana beta 1 tubulin, identifies a single electrophoretically separable beta-tubulin on 2-D-gel Western blots of total protein extracts from A. thaliana seedlings. We show that AABI crossreacts with two of the eight polyglutamylated beta-tubulin isoforms present in purified Nicotiana tabacum tubulin fractionated by high-resolution isoelectric focussing. Immunolocalisation studies using AAB1 revealed that the two N. tabacum polyglutamylated beta 1-tubulin isoforms are utilised in all four plant microtubule arrays (the interphase cortical array, the preprophase band, the spindle and the phragmoplast) indicating that there is no apparent subcellular sorting of these isotypes.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.