sj-docx-1-nnr-10.1177_15459683231170539 – Supplemental material for Effects of Neurofeedback on Cognitive Function, Productive Activity, and Quality of Life in Patients With Traumatic Brain Injury: A Randomized Controlled Trial

Supplemental material, sj-docx-1-nnr-10.1177_15459683231170539 for Effects of Neurofeedback on Cognitive Function, Productive Activity, and Quality of Life in Patients With Traumatic Brain Injury: A Randomized Controlled Trial by Pin-Yuan Chen, I-Chang Su, Chun-Ying Shih, Yen-Chun Liu, Yu-Kai Su, Li Wei, Hui-Tzung Luh, Hui-Chuan Huang, Pei-Shan Tsai, Yen-Chun Fan and Hsiao-Yean Chiu in Neurorehabilitation and Neural Repair</p

A Nucleolus-Predominant <i>piggyBac</i> Transposase, NP-mPB, Mediates Elevated Transposition Efficiency in Mammalian Cells

<div><p><i>PiggyBac</i> is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of <i>piggyBac</i> transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3–4 fold increase in <i>piggyBac</i> transposition efficiency is reproducibly observed in mouse and human cells.</p></div

NP-mPB expression has little effect on cell viability.

<p>To probe the potential cytotoxicity of mPB and NP-mPB expression, HEK293T cells were transfected with different amounts of the mPB, NP-mPB, mPB-tGFP, and NP-mPB-tGFP expression vectors. The relative cell viability was evaluated by the MTS assay. There was no significant difference among the different protein-expressing vectors. n  = 3 for each condition; bars indicate mean ± standard error; <i>P</i>>0.05.</p

Subcellular localization of mPB and NP-mPB.

<p>Hela cells were transfected with mPB-tGFP or NP-mPB-tGFP expression vectors and immunostained with an anti-cyclin T1 antibody (red) and an anti-fibrillarin antibody (blue). (A) Fluorescence microscopy revealed that mPB was localized in the nucleus, but largely outside the nucleoli, as indicated by the fibrillarin. The cyclin T1 signal was dotted and spared the nucleus. (B) In contrast, NP-mPB was localized in the nucleus, with a strong staining in the nucleoli. There was a dotted pattern of NP-mPB in the nucleus beside the nucleoli. Some of the dots were overlapped with the cyclin T1 signal.</p

Evaluation of the transposition efficiency mediated by the gradient concentrations of PBase.

<p>(A) The NP-mPB group demonstrated more colonies compared to the mPB group in HEK293T cells transfected with varying amounts of plasmid DNA. (B) Colony numbers were quantified, revealing a 2.5- to 6-fold increase in transposon integration efficiency for NP-mPB. The transposition efficiency was higher when more plasmid was transfected for both groups. No overproduction inhibition was noted. n  = 3 for each condition; bars indicate mean ± standard error; ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Insertion site analysis of NP-mPB assisted transposition events.

<p>The lowercase letters indicate the unmatched nucleotides in the flanking sequence alignment.</p

The mPB and NP-mPB protein expression profiles in HEK293T cells transfected with <i>pTriEx-mPB</i> and <i>pTriEx-NP-mPB</i> plasmids.

<p>(A) At 48 h after transfection, the transposase protein level in the <i>pTriEx-mPB</i> group was higher than that in the <i>pTriEx-NP-mPB</i> group. <i>UGm</i> plasmid was cotransfected as a control for transfection efficiency (anti-GFP). <i>pTriEX-HTNC</i> plasmid was transfected as a positive control for anti-His tag antibody (Cre). (B) Western blot analysis of nucleo-cytoplasmic separated lysates revealed that the mPB protein was distributed preferentially in the nucleus and the cytoplasm, whereas almost all NP-mPB protein was localized in the nucleus.</p

Schematic representation of expression constructs for PBase variants, PBase fusion proteins, and the dual fluorescent <i>UGm</i> transposon used in this study.

<p>(A) The mPB and NP-mPB coding sequences of mouse codon-optimized PBase were cloned into the <i>pTriEx-HTNC</i> plasmid by replacing the <i>Cre</i> cassette between <i>Spe</i>I and <i>Xho</i>I. The mPB and NP-mPB coding sequences were preceded by a hexa-histidine encoding sequence (6× His tag). NP-mPB included an additional nucleolus-predominant (NP) signal peptide. (B) The <i>pTriEx-mPB-tGFP</i> and <i>pTriEx-NP-mPB-tGFP</i> constructs expressed tGFP-fused PBases. The tGFP moiety allowed real-time imaging of the PBase variant subcellular distributions. The <i>pTriEx-mPB-2A-eGFP</i> and <i>pTriEx-NP-mPB-2A-eGFP</i> plasmids expressed PBase variants, which were linked to eGFP by the self-cleaving 2A peptide. All protein-encoding cassettes were transcriptionally regulated by a hybrid promoter composed of the CMV immediate early enhancer fused to the chicken β-actin promoter (CAG), and followed by a polyadenylation signal sequence (pA). (C) Flanking the 5′ and 3′ inverted terminal repeats (ITRs) (empty arrows), the dual fluorescent transposon (<i>pXL-T3-Neo-UGm-cHS4X</i>; <i>UGm</i>) carried a human ubiquitin C (UBC) promoter-driven <i>H2B-eGFP-2A-mCherry-GPI</i> (Gm) transgene that labeled the transposed cells with a characteristic chromatin EGFP and membrane mCherry dual fluorescence. Additional abbreviations: Neo^r, a neomycin phosphotransferase expression cassette providing resistance to G418 selection; 2× Ins, two copies of the insulator sequence from chicken β-globin.</p

The NP-mPB showed a 3- to 4-fold increase in transposon integration efficiency.

<p>The <i>UGm</i> transposon, alone or mixed with the mPB or NP-mPB expression vector, was co-electroporated into mouse or human embryonic stem (ES) cells and then selected for G418 resistance. Surviving colonies were stained by crystal violet and counted. There were more colonies in the NP-mPB group than in the mPB group in mouse ES (A), human ES (B), and Hela cells (C). (D–F) Transposition events were quantified by counting the colonies on culture plates. The transposition efficiency mediated by NP-mPB was increased 3- to 4-fold in mouse ES cells (D), 3-fold in human ES cells (E), and approximately 3-fold in Hela cells (F). n  = 3 for each condition; bars indicate mean ± standard error; ** <i>P</i><0.01, *** <i>P</i><0.001.</p

<em>R26R-GR</em>: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

<div><p>Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a <em>R26</em> dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.</p> </div