Potential Therapeutic Effects of Meditation for Treating Affective Dysregulation

Affective dysregulation is at the root of many psychopathologies, including stress induced disorders, anxiety disorders, and depression. The root of these disorders appears to be an attenuated, top-down cognitive control from the prefrontal cortices over the maladaptive subcortical emotional processing. A form of mental training, long-term meditation practice can trigger meditation-specific neuroplastic changes in the brain regions underlying cognitive control and affective regulation, suggesting that meditation can act as a kind of mental exercise to foster affective regulation and possibly a cost-effective intervention in mood disorders. Increasing research has suggested that the cultivation of awareness and acceptance along with a nonjudgmental attitude via meditation promotes adaptive affective regulation. This review examined the concepts of affective regulation and meditation and discussed behavioral and neural evidence of the potential clinical application of meditation. Lately, there has been a growing trend toward incorporating the “mindfulness” component into existing psychotherapeutic treatment. Promising results have been observed thus far. Future studies may consider exploring the possibility of integrating the element of “compassion” into current psychotherapeutic approaches

β-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy

The Locus Control Region (LCR) requires intronic elements within b-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional bglobin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igm 39MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors

Rapid Transcriptional Pulsing Dynamics of High Expressing Retroviral Transgenes in Embryonic Stem Cells

Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Visualization of transcription foci.

<p>A. Transcription foci were detected as focal dots (yellow arrows) in Clone B6 and 3A10 after transient transfection of MS2-EGFP. Uninfected J1 ES cells were used as a control. Scale bar = 2.5 µM. B. Sample images from multiple z-stacks of a cell from Clone 3A10 displaying a focal dot over 6 consecutive focal planes. Z-stacks were 0.3 µM apart. Scale bar = 5 µM. C. Quantification of the percentage of cells with transcription foci in EGFP-positive cells. (n = 2; at least 47 cells were examined for each n).</p

Transcription dynamics of Clone 3A10.

<p>A. Representative detection of transcriptional pulses in one cell from Clone 3A10 at 2.5 minute intervals. Transcription foci indicated by arrows. Scale bar = 2.5 µM. B. Transcription foci of the cell depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037130#pone-0037130-g004" target="_blank">Figure 4A</a> was quantified for EGFP intensity over time. EGFP intensity plots were determined by subtracting background fluorescence at each time point (Blue line). Red line represents visual scoring of inferred transcriptional activity. C. Transcriptional activity of cells possessing EGFP foci at the start of live imaging. D. Summary of transcriptional dynamics displayed by all pulsing cells in Clone 3A10. Green squares indicate timepoints with detectable transcription foci and gray squares represent timepoints without transcription foci. Each square represents 2.5 minutes. The cell shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037130#pone-0037130-g004" target="_blank">Figure 4A</a> is denoted with an asterisk. E. Representative image of transcriptional pulsing of a Clone 3A10 cell recorded at 30 sec intervals. Scale bar = 2.5 µM. F. Intensity time series data (blue line) and visual scoring of inferred transcriptional activity (red line) on the pulsing cell depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037130#pone-0037130-g004" target="_blank">Figure 4E</a>. G. Cumulative periods of transcriptional activity recorded by the two image intervals. 60 cells were examined for 2.5 minutes interval and 12 cells were examined for 30 seconds interval.</p

Detection of rapid transcriptional pulsing in Clone B6.

<p>A. Rapid and long transcriptional pulses (arrow) detected in a representative cell of Clone B6. Image acquisition was performed every 27 seconds. Scale bar = 2.5 µM. B. Intensity time series data (blue line) of the pulsing cell depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037130#pone-0037130-g005" target="_blank">Figure 5A</a>. Red line represents inferred transcriptional activity by visual scoring.</p

Focal doublets on sister chromatids associate with transcription factories.

<p>A. Transcription doublets can be seen in a cell that has undergone replication at the integration site. The pair of transcription foci (green) overlaps the replicated DNA FISH signals (red) on one pair of alleles. The nucleus is counterstained with DAPI (cyan). B. ImmunoFISH of Clone B6 focal doublets (green) show they associate with distinct RNA Pol II factories (violet). A single focal plane with focal doublets is shown. The nucleus is counterstained with DAPI (cyan). Scale bar = 2.5 µM. C. Detection of transcriptional doublets in Clone 3A10 persisting through 10 minutes of image acquisition. Scale bar = 2.5 µM. D. Dynamics of transcription doublets in Clone B6. Scale bar = 2.5 µM.</p

Development of the MS2 system to detect transcription sites of a retroviral transgene.

<p>A. Schematic diagram for detecting transcription sites from retroviral transgenes. The MS2 stem-loop was inserted into a HSC1 retrovirus backbone expressing mRFP. The structure of provirus after integration into the genome is shown. Location of restriction enzymes digestion sites, probe used for southern blot analysis (gray box) and primers for PCR (red arrows) to confirm size of stem-loops are indicated. B. Southern blot analysis of genomic DNA from Clone B6 and 3A10 of infected ES cells digested with various enzymes. (E = <i>Eco</i>RI, B = <i>Bam</i>HI, H = <i>Hind</i>III, S = <i>Spe</i>I, N = <i>Nhe</i>I) Digested DNA was hybridized to the mRFP probe. No <i>Hind</i>III site is found within the provirus. C. PCR analysis of number of stem-loops integrated into the genome in the two clones (top) and the number of stem-loops transcribed from each clone (bottom). Uninfected J1 ES cells were used as a control. Actin was used as a loading control. D. Flow cytometry histograms showing expression of mRFP in Clone B6 (top) and Clone 3A10 (bottom). Red line denotes uninfected control J1 cells and blue line indicates clone being interrogated.</p