We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, ...including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome ...organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30–600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.
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•CRISPR-GO is a versatile system for targeting genome loci to nuclear compartments•An inducible and reversible approach to study spatial genome organization•CRISPR-GO mediates rapid and reversible de novo Cajal body (CB) formation•CB-chromatin colocalization represses distal (30–600 kb) gene expression
An engineered CRISPR-based platform for inducible recruitment of specific genomic loci to distinct nuclear compartments reveals positional effects on gene expression and cellular function.
Despite the mortality benefit of cardiac rehabilitation (CR) participation, as well as its cost-effectiveness for people with peripheral artery disease (PAD), there are limited data on adherence and ...completion of CR in those with and without concomitant coronary artery disease (CAD). The objective of this study was to compare CR pre-participation withdrawal and noncompletion between patients with PAD and concomitant PAD and CAD (PAD/CAD) versus matched and unmatched patients with CAD (uCAD).
Consecutively referred patients between 2006-2017 with PAD (n = 271) and PAD/CAD (n = 610) were matched to CAD by age, sex, diabetes, smoking status, and referral year. The uCAD (n = 14 487) group was included for comparison. Reasons for withdrawal were ascertained by interview.
There were no significant differences in pre-participation withdrawal between PAD and matched CAD (46 vs 43%, P = .49), nor in noncompletion (22 vs 18%, P = .28). Results were similar for PAD/CAD and matched CAD (withdrawal: 36 vs 34%, P = .37) and (noncompletion: 25 vs 23%, P = .46). A smaller proportion of patients with uCAD withdrew (28%) than patients with PAD ( P < .001) and PAD/CAD ( P < .001), with no difference in noncompletion ( P > .40, both). There were no differences between PAD and PAD/CAD and their matched counterparts for medical and nonmedical reasons for withdrawal and noncompletion ( P ≥ .25, all).
Pre-participation withdrawal rates were similar between patients with PAD, PAD/CAD, and their matched cohorts but greater than patients with uCAD. Once patients started CR, there were similar completion rates among all groups. Reports that patients with PAD are less likely to start CR may be related to their complex medical profile rather than PAD alone. Strategies to improve participation among patients with PAD should focus on the immediate post-referral period.
Cholesterol is an essential structural component of all membranes of mammalian cells where it plays a fundamental role not only in cellular architecture, but also, for example, in signaling pathway ...transduction, endocytosis process, receptor functioning and recycling, or cytoskeleton remodeling. Consequently, intracellular cholesterol concentrations are tightly regulated by complex processes, including cholesterol synthesis, uptake from circulating lipoproteins, lipid transfer to these lipoproteins, esterification, and metabolization into oxysterols that are intermediates for bile acids. Oxysterols have been considered for long time as sterol waste products, but a large body of evidence has clearly demonstrated that they play key roles in central nervous system functioning, immune cell response, cell death, or migration and are involved in age-related diseases, cancers, autoimmunity, or neurological disorders. Among all the existing oxysterols, this review summarizes basic as well as recent knowledge on 25-hydroxycholesterol which is mainly produced during inflammatory or infectious situations and that in turn contributes to immune response, central nervous system disorders, atherosclerosis, macular degeneration, or cancer development. Effects of its metabolite 7α,25-dihydroxycholesterol are also presented and discussed.
High blood pressure (BP) variability after endovascular stroke therapy is associated with poor outcome. Conventional BP variability measures require long recordings, limiting their utility as a risk ...assessment tool to guide clinical decision-making. Here, we performed rapid assessment of BP variability by spectral analysis and evaluated its association with early clinical improvement and long-term functional outcomes.
We conducted a prospective study of 146 patients with anterior circulation ischemic stroke who underwent successful endovascular stroke therapy. Spectral analysis of 5-minute recordings of beat-to-beat BP was used to quantify BP variability. Outcomes included initial clinical response and modified Rankin Scale at 90 days.
Increased BP variability at high frequencies was independently associated with poor functional outcome at 90 days (adjusted odds ratio aOR, 1.85 95% CI, 1.07-3.25,
=0.03; low-/high-frequency ratio aOR, 0.67 95% CI, 0.46-0.92,
=0.02) and reduced likelihood of an early neurological recovery (aOR, 0.62 95% CI, 0.44-0.91,
=0.01 and aOR, 1.37 95% CI, 1.03-1.87,
=0.04, respectively).
High-frequency BP oscillations after successful reperfusion may be harmful and associate with a decreased likelihood of neurological recovery and favorable functional outcomes. Rapid assessment of BP variability throughout the postreperfusion period is feasible and may allow for a more personalized BP management.
Cardiac rehabilitation (CR) is recommended for patients with coronary (CAD) and peripheral (PAD) artery disease. However, no study has compared changes in cardiorespiratory fitness (VO2peak) or ...exercise prescription progression among PAD, CAD, and concomitant PAD and CAD (BOTH). The objectives of this study were to 1) compare change in VO2peak among patients with PAD, CAD, and BOTH, and 2) examine progression in exercise prescription parameters in a comprehensive 6-month cardiac rehabilitation (CR) program.
A retrospective analysis of patient data recorded from 2006 to 2017 from a large urban hospital was conducted. Patients with PAD (n = 63) and BOTH (n = 164) were included in the analyses. Patients with CAD (n = 63) were matched to PAD by sex (36.5% female), age (69 years), smoking status, diabetes, and year in program.
There were significant improvements in VO2peak from baseline to 6 months in all groups (CAD +2.7 ± 3.4 mL⋅kg−1⋅min−1, PAD +2.4 ± 3.8 mL⋅kg−1⋅min−1, BOTH +1.8 ± 3.1 mL⋅kg−1⋅min−1; all P < 0.001). Between-group differences were significant between PAD and CAD as well as between CAD and BOTH (P = 0.001). Walking distance, duration, and pace increased for all groups over 6 months (P < 0.001), with a significant difference in pace between CAD and BOTH (P = 0.006).
Patients with PAD, CAD, and BOTH had significant improvements in VO2peak following a 6-month CR program. However, despite similar prescribed walking distance and duration, improvements in VO2peak were mitigated in PAD and BOTH compared with CAD. These results support benefits of CR for patients diagnosed with PAD, but alternate exercise strategies should be explored for patients with PAD.
La réadaptation cardiaque (RC) est recommandée aux patients atteints de maladie coronarienne (MC) et de maladie artérielle périphérique (MAP). Toutefois, aucune étude n’a permis de comparer les changements dans la forme cardiorespiratoire (VO2max) ou la progression des paramètres de la prescription d’exercice des patients atteints de MAP, de MC, ou d’une MAP et d’une MC concomitantes (les deux maladies). Les objectifs de la présente étude étaient de : 1) comparer les changements entre la VO2max des patients atteints de MAP, de MC et des deux maladies; 2) examiner la progression des paramètres de la prescription d’exercice d’un programme complet de RC de six mois.
Nous avons mené une analyse rétrospective des données consignées des patients de 2006 à 2017 d’un grand hôpital en milieu urbain. Dans les analyses, nous avons inclus les patients atteints de MAP (n = 63) et des deux maladies (n = 164). Nous avons apparié les données des patients atteints de MC (n = 63) aux données des patients atteints de MAP du programme selon le sexe (36,5 % de femmes), l’âge (69 ans), la consommation de tabac, le diabète et l’année.
Nous avons observé des améliorations significatives de la VO2max entre le début et après six mois dans tous les groupes (MC + 2,7 ± 3,4 ml/kg−1 min−1, MAP + 2,4 ± 3,8 ml/kg−1 min−1, les deux maladies + 1,8 ± 3,1 ml/kg−1⋅min−1; tous P < 0,001). Les différences entre les groupes étaient significatives entre les patients atteints de MAP et les patients atteints de MC ainsi qu’entre les patients atteints de MC et des deux maladies (P = 0,001). La distance, la durée et la vitesse de marche augmentaient dans tous les groupes au cours des six mois (P < 0,001), mais la vitesse de marche montrait une différence significative entre les patients atteints de MC et les patients atteints des deux maladies (P = 0,006).
Les patients atteints de MAP, de MC et des deux maladies montraient des améliorations significatives de la VO2max après avoir réalisé un programme de RC de six mois. Toutefois, malgré la prescription d’une marche de distance et de durée similaires, les améliorations de la VO2max étaient moins importantes chez les patients atteints de MAP et des deux maladies que chez les patients atteints de MC. Ces résultats corroborent les avantages de la RC pour les patients ayant un diagnostic de MAP, mais d’autres stratégies d’exercice devraient faire l’objet d’études auprès de ces patients.
Increasing evidence suggests that gonadotropin-releasing hormone (GnRH), corazonin, adipokinetic hormone (AKH), and red pigment-concentrating hormone all share common ancestry to form a GnRH ...superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates.
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a ...sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
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