Real-world keystroke dynamics are a potentially valid biomarker for clinical disability in multiple sclerosis

Background: Clinical measures in multiple sclerosis (MS) face limitations that may be overcome by utilising smartphone keyboard interactions acquired continuously and remotely during regular typing. Objective: The aim of this study was to determine the reliability and validity of keystroke dynamics to assess clinical aspects of MS. Methods: In total, 102 MS patients and 24 controls were included in this observational study. Keyboard interactions were obtained with the Neurokeys keyboard app. Eight timing-related keystroke features were assessed for reliability with intraclass correlation coefficients (ICCs); construct validity by analysing group differences (in fatigue, gadolinium-enhancing lesions on magnetic resonance imaging (MRI), and patients vs controls); and concurrent validity by correlating with disability measures. Results: Reliability was moderate in two (ICC = 0.601 and 0.742) and good to excellent in the remaining six features (ICC = 0.760–0.965). Patients had significantly higher keystroke latencies than controls. Latency between key presses correlated the highest with Expanded Disability Status Scale (r = 0.407) and latency between key releases with Nine-Hole Peg Test and Symbol Digit Modalities Test (ρ = 0.503 and r = −0.553, respectively), ps < 0.001. Conclusion: Keystroke dynamics were reliable, distinguished patients and controls, and were associated with clinical disability measures. Consequently, keystroke dynamics are a promising valid surrogate marker for clinical disability in MS

Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (∼10−6 to 10−8), its practical application has been largely restricted to specific experimental systems that allow selection of the few cells that become genetically modified. HR-mediated gene targeting has nonetheless revolutionized genetics by greatly facilitating the analysis of mammalian gene function. Recent studies showed that generation of double-strand DNA breaks at specific loci by designed endonucleases greatly increases the rate of homology-directed gene repair. These findings opened new perspectives for HR-based genome editing in higher eukaryotes. Here, we demonstrate by using donor DNA templates together with the adeno-associated virus (AAV) Rep78 and Rep68 proteins that sequence- and strand-specific cleavage at a native, predefined, human locus can also greatly enhance homology-directed gene targeting. Our findings argue for the development of other strategies besides direct induction of double-strand chromosomal breaks to achieve efficient and heritable targeted genetic modification of cells and organisms. Finally, harnessing the cellular HR pathway through Rep-mediated nicking expands the range of strategies that make use of AAV elements to bring about stable genetic modification of human cells

Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy

Characterization of Parameters Required for Effective Use of Tamoxifen-Regulated Recombination

Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population

Development of a Chromosomally Integrated Metabolite-Inducible Leu3p-α-IPM “Off-On” Gene Switch

Background: Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression. Methodology/Principal Findings: Here, we show that a chromosomally integrated yeast ‘Leu3p-a-IRM ’ system constitutes a ligand-inducible regulatory ‘‘off-on’ ’ genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of a-isopropylmalate (a-IRM) in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable a-IPM presents an EC 50 equal to 0.8837 mM and fast ‘‘OFF-ON’ ’ kinetics (t 50ON = 43 min, t 50OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo. Conclusions/Significance: Our results demonstrate that the ‘Leu3p-a-IRM ’ constitutes a simpler and safer system for inducible gene expression in biomedical applications