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Blood vessels in the central nervous system (CNS) form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells to peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizures, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB-dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders.

Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the β-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for β-hemoglobinopathy therapeutic intervention. The NuRD chromatin complex participates in γ-globin repression. Here we use pooled CRISPR screening to comprehensively disrupt NuRD protein coding sequences in human adult erythroid precursors. We find essential for fetal hemoglobin (HbF) control a nonredundant subcomplex of NuRD protein family paralogs, whose composition we corroborate by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identifies key protein interfaces where in-frame alleles result in loss-of-function due to destabilization or altered function of subunits. We ascertain mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrate that sequestering CHD4 from NuRD phenocopies these mutations. This work indicates a generalizable approach to discover protein complex features amenable to rational biochemical targeting., Editorial summary: Comprehensive CRISPR mutagenesis targeting all members of the NuRD complex identifies a specific sub-complex required for fetal globin silencing and informs a rational targeting strategy for elevating globin levels while avoiding cytotoxicity.

Ein mannlicher 72 jahriger Patient mit Pra-Sezary Syndrom, der auf PUVA nicht ansprach, wurde systemisch mit einer low-dose Methotrexat (MTX)-Therapie (12,5 mg bis 25 mg/ Woche i. v.) behandelt. Das klinische Bild mit Erythrodermie, Lichenifikation, Schuppung, odematoser Schwellung der Extremitaten und einem qualenden Juckreiz besserte sich durch MTX rasch. Parallel dazu lies sich ein Ruckgang der Eosinophilie im peripheren Blut feststellen. Alle ubrigen hamatologischen Parameter incl. flowzytometrisch analysierter T-Zell-Subsets blieben im Normbereich. Eine Immunsuppression trat nicht auf. Der Patient tolerierte die MTX-Therapie subjektiv sehr gut, objektive Nebenwirkungen wurden nicht beobachtet. Nach Erzielung der kompletten Remission wurde die MTX-Therapie abgesetzt. In der Nachsorgephase von 27 Monaten trat bislang kein Rezidiv auf. Anhand unserer Kasuistik scheint die low-dose MTX-Therapie im Fruhstadium des Sezary-Syndroms (Pra-Sezary Syndrom) eine ausgezeichnete Behandlungsstrategie zu sein, was anhand groserer Patientenkollektive systematisch uberpruft werden muste.

The development of advanced railway vehicles requires an efficient modelling and simulation of wheel-rail systems under complex conditions and maneuvers. For that reason, the multibody system simulation software SIMPACK was enhanced recently with a generally applicable wheel-rail contact module to be outlined in this paper. This module comprises the possibility of applying a constrained or an elastic contact model, single or multiple contact locations per wheel-rail element, analytic or measured track geometries including irregularities and rail profiles, varying in longitudinal direction (switches). A simulation study of a passenger car running through a switch demonstrates the efficiency of the constrained wheel-rail contact model extended for the simulation of multiple contacts. As a second example a freight train is investigated and its simulation results are compared to measurements in order to validate the simulation tool as well as the model.

Quasielastic gamma‐ray scattering of 46.5‐keV Mossbauer photons by polydimethylsiloxane has been studied at room temperature as a function of dilution in benzene. The high energy resolution of this novel technique allowed the separation of the scattering signal into a narrow component associated with stiff motions along the polymer chain backbone and a quasielastic component associated with softer side group motions. The narrow component disappears upon dilution in benzene while the intensity of the quasielastic component grows proportionately. This result is interpreted as a softening of the backbone normal modes upon dilution.

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)