An Elisa-Based Platform for Rapid Identification of Structure-Dependent Nucleic Acid-Protein Interactions Detects Novel DNA Triplex Interactors

Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)-mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure-protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide-induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair

How are base excision DNA repair pathways deployed in vivo? [version 1; referees: 4 approved]

Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them

BALTA Project C6 - From Social Economy to Solidarity Economy: Changing Perspectives in a Volatile World (Phase Two)

Phase One of this research developed the theoretical conceptual analysis comparing the social economy and solidarity economy concepts.This proposal/plan describes a second phase research project for the BC-Alberta Social Economy Research Alliance (BALTA) to explore the relative merits and limitations of social economy and solidarity economy conceptualizations by applying both to case studies of the Chicago Manufacturing Renaissance and RESO (Regroupement pour la Relance Économique et Sociale du Sud-Ouest) in Montreal, Quebec.BC-Alberta Social Economy Research Alliance (BALTA) ; Social Sciences and Humanities Research Council of Canada (SSHRC) ; Canadian Centre for Community Renewal (CCCR) ; Center for Labor and Community Researc

Cell markers staining of spinal cord cryosections from ²⁸Si exposed rats.

<p>(A) Left image: Spinal cords from rats exposed to 0.5 Gy of 300 MeV/n ²⁸Si,were isolated after 6 months post exposure. 8 μm thick cryosections from T7 (dorso cortico spinal (dcs) ventral column white matter section staining at 40X magnification is shown here) stained for APE1 and cell markers (A2B5, NG2), and imaged on Zeiss Axiovert Imaging system as described in Materials and Methods. Right image: Quantification of staining shown in left image. Total % of positive A2B5, NG2 and APE1 stains were measured and divided by the number of nuclei present in each field of view (n = 1 for one view). We averaged trend of staining per field of view and then averaged all the field of views. For those images we had n = 4 for all conditions except F1 where we had n = 3. The * represents p < 0.05 compared to the control. Other than that there was no significance between the conditions (although a trend is there). The scoring was obtained as an average of two independent investigators in a blinded fashion to nullify any bias which arise from just one investigator’s analysis. (B) Duplicate ventral column white matter section from Fig 2 samples, fixed and stained for APE1 and GFAP, imaged on FM at 40X magnification is shown here. (C) APE1 endonuclease activity measured from T7 sections of ²⁸Si exposed rats’ spinal cords, 6 months post exposure by specific endonuclease assay described in methods. Increase in progenitor cell, immature OL and astrocytes seen up to two fractions, where as APE1 decreased with fractionation.</p

Cognition testing of control, protons (1 Gy of 250 MeV protons) (P-irr) and HZE exposed (0.5 Gy Fe/ Si) rats was performed at 6 months of post exposure.

<p>T test showed a significant difference between the Fe irr versus control group (p<0.002). 10 rats were analyzed per fraction, with 40 rats per ion (30 + 10 controls).</p

Novel Object Recognition Testing for low LET radiation exposed rats: Cognition testing for control, protons-irradiated (1 Gy of 250 MeV protons) (P-irr) and X-rays irradiated (1Gy X-rays) (x-irr) rats were performed at 9 months post exposure.

<p>Bars represent means and standard errors of the percentage of time spent by rats investigating a new object in the presence of a familiar one during a 3 min testing period. Memory function percentage revealed significant effect of exposure (p<0.05).</p

TSPO quantification in forebrain of X-rays and protons exposed (only at their thoracic spinal columns) rats: Results are mean +/- SE (Standard Error) of TSPO specific binding, expressed in nCi/mg in various brain regions of rat in control (n = 3), Proton irr = 3 and X-rays- irr = 3 rat brain regions after nine months of exposure to X-rays and protons to their thoracic spinal columns.

<p>Non-specific binding assessed in the presence of excess unlabeled PK11195 was subtracted from all measurements. Two ways ANOVA reveals highly significant effect between the group (p<0.0001) and of region (p<0.0069). Subsequent post hoc analyses were performed using Fisher's PLSD test.</p