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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.

SUMMARYTraumatic spinal cord injury (SCI) produces a complex syndrome that is expressed across multiple endpoints ranging from molecular and cellular changes to functional behavioral deficits. Effective therapeutic strategies for CNS injury are therefore likely to manifest multi-factorial effects across a broad range of biological and functional outcome measures. Thus, multivariate analytic approaches are needed to capture the linkage between biological and neurobehavioral outcomes. Injury-induced neuroinflammation (NI) presents a particularly challenging therapeutic target, since NI is involved in both degeneration and repair1,2. Here, we used big-data integration and large-scale analytics to examine a large dataset of preclinical efficacy tests combining 5 different blinded, fully counter-balanced treatment trials for different acute anti-inflammatory treatments for cervical spinal cord injury in rats. Multi-dimensional discovery, using topological data analysis3 (TDA) and principal components analysis (PCA) revealed that only one showed consistent multidimensional syndromic benefit: intrathecal application of recombinant soluble TNFα receptor 1 (sTNFR1), which showed an inverse-U dose response efficacy. Using the optimal acute dose, we showed that clinically-relevant 90 min delayed treatment profoundly affected multiple biological indices of NI in the first 48 hrs after injury, including reduction in pro-inflammatory cytokines and gene expression of a coherent complex of acute inflammatory mediators and receptors. Further, a 90 min delayed bolus dose of sTNFR1 reduced the expression of NI markers in the chronic perilesional spinal cord, and consistently improved neurological function over 6 weeks post SCI. These results provide validation of a novel strategy for precision preclinical drug discovery that is likely to improve translation in the difficult landscape of CNS trauma, and confirm the importance of TNFα signaling as a therapeutic target.

This study demonstrates how gene expression profiles of white blood cells in acute spinal cord injury patients can be utilized for the diagnosis of the injury severity and the neurological level of injury., Diagnosis of spinal cord injury (SCI) severity at the ultra-acute stage is of great importance for emergency clinical care of patients as well as for potential enrollment into clinical trials. The lack of a diagnostic biomarker for SCI has played a major role in the poor results of clinical trials. We analyzed global gene expression in peripheral white blood cells during the acute injury phase and identified 197 genes whose expression changed after SCI compared with healthy and trauma controls and in direct relation to SCI severity. Unsupervised coexpression network analysis identified several gene modules that predicted injury severity (AIS grades) with an overall accuracy of 72.7% and included signatures of immune cell subtypes. Specifically, for complete SCIs (AIS A), ROC analysis showed impressive specificity and sensitivity (AUC: 0.865). Similar precision was also shown for AIS D SCIs (AUC: 0.938). Our findings indicate that global transcriptomic changes in peripheral blood cells have diagnostic and potentially prognostic value for SCI severity.

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.

Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and selection of therapy-resistant clones. While genomic alterations are known contributors to ITH, emerging studies highlight functional roles for epigenomic ITH which integrates differentiation status, stochastic events, and microenvironmental inputs. Here, we have established a novel platform for integrative characterization of genomic and epigenomic ITH of glioblastoma in three-dimensional (3-D) space. In collaboration with neurosurgeons and biomedical imaging experts, we utilize 3-D surgical neuro-navigation to safely acquire ~10 tumor samples per patient representing maximal anatomical diversity. We conduct whole-exome sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-Seq) on each sample. The spatial location of each sample is mapped by its 3-D coordinates, allowing 360-degree visualization of genomic and epigenomic ITH for each patient. We demonstrate this approach on 8 patients with primary IDH-WT glioblastoma (83 spatially mapped samples), providing unprecedented insight into their spatial organization at the genomic and epigenomic levels. We link genetically defined tumor subclones to patterns of open chromatin and gene regulation, revealing underlying transcription factor binding at active promoters and enhancers. We also identify ITH in whole-genome doubling and focal oncogene amplification events in multiple patients, which we then link with epigenomic ITH. Further, to study microenvironmental inputs and their contribution to epigenomic ITH, we conduct deconvolution of RNA sequencing and ATAC-Seq data by analyzing feature co-variation. We resolve the 3-D spatial organization of immune, neural, and other nontumor cell types present in glioblastoma, characterizing their functional states and interactions with tumor cells. This work provides the most comprehensive spatial characterization of genomic and epigenomic ITH to date in glioblastoma. As a resource for further investigation, we have developed an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

Adult low-grade gliomas generally progress to glioblastoma, a more aggressive CNS tumor with an extremely poor prognosis. Despite intensive efforts, numerous promising glioma therapies have failed to provide survival benefits. These failures reflect many factors, including intertumoral heterogeneity and immunosuppression by the tumor microenvironment (TME). We propose a novel approach to addresses these challenges through integrative deconvolution of bulk gene expression data generated from more than 5000 human gliomas and 7000 normal human brain samples. Inherent variation in the cellular composition and cellular activities of these samples allowed us to identify highly correlated modules of genes that represent specific cell types and cell states. By comparing gene coexpression modules in glioma vs. normal human brain, we have identified cell type-specific gene expression changes in the glioma TME that are highly reproducible. In contrast to single-cell methods, which sample only a fraction of the tumor tissue and fail to capture major nonmalignant cell-types, our results derive from billions of cells and thousands of individuals and are therefore highly robust. We find that a number of genes encoding cell-surface proteins are specifically up-regulated in immune and vascular cells of the glioma TME. Surprisingly, among those genes up-regulated in glioma vasculature are multiple members of the angiotensin pathway, suggesting non-canonical roles for these proteins in the glioma setting. We propose that these proteins may form a specific ‘zip code’ for glioma within the brain’s vasculature that can be targeted directly or by conjugation with existing drugs. More generally, our analytical approach has revealed reproducible gene expression changes in specific cell types of the glioma TME that provide more stable therapeutic targets than those that are expressed by genetically mutable malignant cells. We have also discovered novel, aberrantly coexpressed genes in microglia, oligodendrocytes, and astrocytes which we are testing in state-of-the-art human brain assembloid systems.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets we have developed CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genome, transcript, and protein coordinates and structure. No available tool has similar functionality. The ensuing genotype-phenotype linear and 3D maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg−1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target., Benzoxaboroles have been shown to be active against different pathogens. Here, the authors show that the benzoxaborole AN3661 inhibits Plasmodium falciparum in vitro and in mouse models, and identify a homologue of a mammalian cleavage and polyadenylation specificity factor as a drug target.

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.

The prime condition for the virtual design of mechatronic railway vehicles is a software environment comprising the different computational engineering tools being interconnected by efficient interfaces. The paper presents such an environment with a multibody system software in its centre and a couple of interfaces to other engineering software tools around. Especially in view of mechatronics, the different types of coupling to control engineering software are of interest. The simulation of railway vehicles calls for a specific module describing, besides others, the highly nonlinear contact between wheel and rail. The state-of-the-art of such a simulation tool chain is illustrated for the complex analysis and design issue of a “Mechatronic Train”.

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.

Using very-high-intensity ({approximately}70 Ci) {sup 183}Ta M{umlt o}ssbauer sources, we have measured the Debye-Waller factors (DWF{close_quote}s) of sodium chloride, potassium chloride, and potassium bromide single crystals for several of the (h00) and (nnn) Bragg reflections. We have used an approach which properly accounts for thermal expansion over the temperature range of our experiment, from 90 K to 900 K, about 100 K below the melting point of our crystals. We have found that a procedure used to analyze data by earlier workers leads to incorrect parameters in the Debye-Waller factor exponent, and our procedure does not require empirical parameters to account for the effects of thermal expansion. Additionally, we find three items of significance. Contrary to earlier results, we observe that the cations and the anions have identical DWF{close_quote}s in NaCl and also in KBr. We observe terms in the expansion of the DWF exponential which are quartic in the scattering wave vector {rvec Q} in NaCl and KCl, with some evidence for a Q{sup 4} term in KBr. The size of the Q{sup 4} contribution is reported and varies with the direction of momentum transfer. We also observe that the Debye temperature and the coefficient of the anharmonic Q{supmore » 2} term also vary with the direction of momentum transfer. We believe our data are the definitive evidence for a nonspherical thermal cloud in a cubic crystal; the ions have a larger amplitude of oscillation in the [h00] direction than in the [nnn] direction, contrary to the commonly held view of crystallographers that the most general form of the mean-square thermal motion is of an ellipsoid shape. {copyright} {ital 1998} {ital The American Physical Society}« less

We have used 70 Ci 183Ta Mossbauer sources to measure the Debye--Waller factors of NaCl, KCl, and KBr single crystals for several of the (h00) and (nnn) Bragg reflections. We have found that the procedure used to analyze data by earlier workers leads to incorrect parameters in the Debye--Waller factor exponent, and we use a procedure that eliminates the need to introduce an empirical parameter to account for thermal expansion effects. We observe that both the cations and anions in NaCl and KBr have identical Debye--Waller factors in the [nnn] direction. We observe Q4 terms in NaCl and KCl, with some evidence for a Q4 term in KBr. The size of this contribution varies with the direction of momentum transfer and will be reported. We observe that the Debye temperature and the coefficient of the anharmonic Q2 term also vary with direction of momentum transfer. We believe this to be the first definitive evidence for a non-spherical thermal cloud in a cubic crystal; the ions have a larger amplitude of oscillation in the [h00] direction than in the [nnn] direction. This effect is temperature dependent and greater at lower temperatures.

Genome-wide association studies (GWASs) have identified thousands of common genetic variants associated with human traits and disease susceptibility. Given that the majority of GWAS-identified SNPs are located within non-coding regions of the genome, the mechanisms of these trait associations are frequently unknown. Here we investigate the biological underpinnings of common trait-associated genetic variation at ATP2B4, a gene on chromosome 1q32 that encodes a major plasma membrane calcium ATPase (also known as PMCA4). Genetic variation within this locus is associated with several phenotypes including various red blood cell traits (mean corpuscular hemoglobin concentration and red cell distribution width) as well as susceptibility to severe malaria infection. We conducted an expression quantitative trait loci (eQTL) mapping analysis in human erythroblasts and identified a set of SNPs associated with ATP2B4 gene expression in cis. These included the same genetic variants found by GWAS to be associated with RBC traits and malaria susceptibility. Furthermore, these SNPs overlap an intronic erythroid DNase I hypersensitive site at ATP2B4. An analysis of the ENCODE database showed that this element was erythroid specific in that it lacked DNase I hypersensitivity in >30 queried non-erythroid cell types. We used the CRISPR/Cas9 genome editing system to delete this element in HUDEP-2 immortalized human erythroid precursor cells. We observed that cells bearing a 927 bp biallelic deletion of this noncoding element displayed near complete loss of ATP2B4 expression (3% residual gene expression), while cells bearing heterozygous deletions showed an intermediate gene expression phenotype. We identified a core element that encompassed 3 of the highly trait associated SNPs and 5 GATA1-binding motifs. Biallelic deletion of this 98 bp core led to 83% reduction in ATP2B4 expression. Disruption of individual GATA1-binding motifs resulted in partial reduction of gene expression, suggesting the contribution of multiple binding sites to appropriate gene expression. Overall, this study suggests that variation within an essential erythroid-specific enhancer of ATP2B4 underlies the association of ATP2B4 with RBC traits and malaria susceptibility. Furthermore these results encourage combined analyses of gene expression, chromatin state, and prospective genetic perturbation as a means to explore the variants, elements and genes responsible for heritable blood phenotypes. Disclosures No relevant conflicts of interest to declare.

We have carried out numerous experiments with supersources having intensities in the 100 Curie range. These sources usually require massive shielding, and are not easily moved to carry out Mossbauer spectroscopy. Several of these sources can be used with microfoil conversion electron (MICE) detectors, but they cannot be moved easily either because of the delicate microfoils used, which ideally have thicknesses less than the range of the internally converted electrons. Here, we describe a technique for doing Mossbauer spectroscopy by oscillating a monochromating crystal parallel to the reciprocal lattice vector of the Bragg reflection, this being used to filter out extraneous photons from the beam. Specifically, an LiF crystal is used in diffraction experiments as a filter to scatter the 46.5-keV Mossbauer gamma rays from183Ta by setting it at the (200) Bragg reflection. In the present measurements, the LiF crystal was mounted in the transmission mode and oscillated with a crank along the scattering vectorQ to produce the velocity modulation, with the source, sample and absorber all at rest. The velocity components of the filtering crystal along the incident and scattered beams cause the measured linewidth to be equal to the usual Mossbauer width divided by 2 sinθ, whereθ is the Bragg angle. Measured widths for the (200), (400), (600) and (800) Bragg reflections agreed with the calculated values of 12.08, 6.04, 4.03 and 3.02 cm/s, within our experimental uncertainties. The technique could have applications not only to MICE detectors, but also to very narrow resonances such as67Zn, where the increased velocities required for small Bragg angles could be an advantage and lead to enhanced resolution.

Using high intensity 70 Ci183Ta Mossbauer sources, which have a negligible source resonance self absorption (SRSA), we have measured the relative elastic scattering fractionF(g, T) and the relative scattering intensities for the (200), (400), and (600) planes of a 6N pure silver single crystal as a function of temperature from 81 to 1211 K. The values ofF(Q, T) denote the relative fraction of incident recoilless gamma-rays that scatter from the crystal elastically. As determined by line-shape analysis of the Mossbauer spectra, theF(Q, T) values are calculated from the ratio of the measured values of the scattered to incident beam recoilless fractions, and are used to correct for thermaldiffuse scattering. The Debye-Waller factors derived from the Bragg intensities have a significant anharmonic contribution above room temperature.

The Mossbauer scattering instrument at the Missouri University Research Reactor (MURR) has been used to study the liquid dynamics of glycerol as a function of temperature. The measurements were made by observing the Mossbauer gamma rays from the 46.5-keV level in183W scattered in the direction corresponding to the first liquid structure peak atQ=1.36 A−1. The widths of the Mossbauer velocity spectra were found to be quasielastically broadened by 3.1 μeV at 24 °C up to 55 μeV at 113 °C. The broadening varied quadratically with temperature above the melting point, but showed a linear dependence when plotted versus the absolute temperature divided by the viscosity. As discussed by Singh and Mullen, this latter dependence is expected in the continuous diffusion limit. The original analysis by Singwi and Sojlander was carried out for a diffusing Mossbauer ion, where the observed cross section is the Fourier transform of the self-correlation function. Our case requires the use of the paircorrelation function. Quantitative calculations as well as further experiments are needed.

We have been able to prepare Mossbauer sources having intensities ranging from 10 to 100 Ci for several isotopes, using the Missouri University Research Reactor Facility, MURR. These sources offer rich opportunities for carrying out precision Mossbauer experiments as well as high collimation scattering experiments. Using such intense sources, we have been able to measure the interference parameter of the 46.5 keV Mossbauer transition to about 1% accuracy. We find it to be about 10% greater than predicted by theory, which may have significant implications for time reversal violation experiments. We have also been able to show that the Bragg scattered recoilless fraction scattered from (200) planes of sodium chloride crystals is 94% of that found for the (600) reflection, even though the integrated intensity of this reflection is more than a factor of 10 less than that of the (200) reflection. We highlight some of the experiments that we have carried out and discuss some of the enticing possibilities for the future with sources in the 10–1000 Ci range. One of these is the possibility of doing Mossbauer spectroscopy with a stationary source and absorber, moving only a monochromating crystal filter along the direction of the reciprocal lattice vector associated with the Bragg reflection being used.

We discuss our experiences with exceptionally intense Mossbauer sources (of order 100 Ci) and their use in making precision determinations of line shape parameters. We review our measurements of interference and recoilless fraction using conventional transmission geometry. Fitting simultaneous sets of data with known constraints greatly improves the efficiency of line-shape determination. Measurements of interference have been made using a two-absorber technique first introduced by Mossbauer and coworkers. Our results indicate that the method can be useful for determining interference, but that incorrect line shape parameters are obtained if powder samples are used which do not comply well with the assumption of thickness uniformity in the absorber.

We have made precise final state interference measurements using very intense M\"ossbauer sources and an exact line-shape analysis. Two transitions in tungsten metal, as well as one in iridium metal and one in terbium oxide, have been studied. The interference parameters 100\ensuremath{\beta} found are -1.3(3) for the 99.1-keV transition in $^{183}\mathrm{W}$, -1.2(1) for the 100.1-keV transition in $^{182}\mathrm{W}$, -0.71(8) for the 129.4-keV transition in $^{191}\mathrm{Ir}$, and -0.48(5) for the 58.0-keV transition in $^{159}\mathrm{Tb}$. All values agreed with theory to within error. Our measurement of 1.16(2) cm/s for the $^{191}\mathrm{Ir}$ linewidth agrees with other M\"ossbauer measurements and shows no indication of hyperfine broadening, but disagrees with some non-M\"ossbauer measurements reported in the literature. The tungsten and terbium transitions also show no signs of broadening or splitting.

We have completed a Mossbauer effect (ME) experiment using two absorbers, one stationary and one Doppler shifted, as a means of enhancing the off-resonant signal, in order to confirm earlier measurements in 183W done using single absorbers. A parameter of particular importance is the interference parameter, β, which is relevant to understanding certain time reversal invariance experiments, and which our previous measurements indicated was larger in magnitude than that predicted by theory. The value for 100β reported here is −0.318 (5), which is in excellent agreement with our earlier value and is larger in magnitude than that predicted by theory.

Using very high intensity $(\ensuremath{\sim}70{Ci)}^{183}\mathrm{Ta}\phantom{\rule{0ex}{0ex}}$ M\"ossbauer sources, we have measured the Debye-Waller factors (DWF's) for the (200), (400), and (600) Bragg reflections of three fcc metals (silver, copper, and lead) as a function of temperature from near 80 K to within about 100 K of their melting temperatures. Our reexamination of earlier measurements on these metals indicated problems with data analysis, and there are serious discrepancies between the present results and those reported earlier by Day, Mullen, and Shukla. We have corrected our data for the effects of thermal expansion, in a manner similar to our earlier paper [Phys. Rev. B 57, 889 (1998)] on alkali halides and reinvestigated the anharmonic contributions to the DWF of these three metals. We have found significant ${Q}^{4}$ contributions to the DWF in both silver and copper, with ${m}_{4}$ values of $1.5(3)\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}13}{\mathrm{\AA{}}}^{4}/{\mathrm{K}}^{3}$ and $4.1(15)\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}14}{\mathrm{\AA{}}}^{4}/{\mathrm{K}}^{3},$ respectively.

We have carried out precise M\"ossbauer line-shape analyses in tungsten and iridium metal using an exact representation of the line shape in transmission. By using exceptionally intense sources (\ensuremath{\sim}70 Ci for $^{183}\mathrm{Ta}$) and carefully chosen constraints between sets of M\"ossbauer-effect spectra, we have been able to make a quantitative test of the theory of final-state effects. The temperature dependence of the recoilless fraction, ${\mathit{f}}_{\mathit{a}}$(T), for $^{183}\mathrm{W}$ in tungsten metal has been determined to about 1% accuracy, which is an order of magnitude better than previous M\"ossbauer or x-ray investigations, between 80 and 1067 K. The Debye model fits our results from 80 through 968 K, with a Debye M\"ossbauer temperature of 336.5 K, when a correction for thermal expansion is included. The recoilless fraction data were used to derive a value of 8.76(10) for the internal conversion coefficient for the 46.5-keV transition in $^{183}\mathrm{W}$, with an approach based on the quasiharmonic approximation to the lattice vibrations, which determines the temperature dependence of the recoilless fraction. The interference parameters, times 100, that we find in this investigation of $^{183}\mathrm{W}$ in tungsten metal (46.5 keV) and $^{191}\mathrm{Ir}$ in iridium metal (129 keV), are -0.317(6) and -0.77(10), respectively. These values are both greater in magnitude than the theoretically calculated values by about 10%, although the $^{191}\mathrm{Ir}$ case has errors large enough that the comparison with theory is inconclusive. These results differ with theory, and would indicate that the theoretical calculations of interference need refinement if they are to be used in studies of time-reversal invariance.

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 analytic description of Mossbauer line shape based on the Fourier-transform method [1] makes it possible to precisely determine the recoil-free fraction,f, of a single absorber over a wide temperature range with a single measurement at each temperature. Most measurements of recoil-free fraction have been done with a Lorentzian representation of the Mossbauer line with semi empirical correction factors of uncertain precision. Our method, based on line shape rather than area, is exact for absorbers of any thickness, limited only by the number of terms taken in the expansion. In fact, our exact line-shape description makes it advantageous to use very thick absorbers. As an example, we report values for an absorber of effective thickness 12. The resonance thickness number of the absorber is an intrinsic parameter, so that a fit yields this quantity directly. The correlations that arise between certain variables may be reduced by fitting, simultaneously and with known constraints, spectra taken with different absorbers or with one absorber at various temperatures. Using this technique, we report precise values forf(T) in tungsten using the 46.5 keV transition in183W atT=297 K and 1067 K; these values are 0.300±0.009 and 0.0123±0.0002, respectively.

Common genetic variation associated with fetal hemoglobin (HbF) level and β-hemoglobin disorder clinical severity marks an erythroid enhancer within the BCL11A gene. The 12 kb intronic enhancer contains three ~1 kb erythroid DNase I hypersensitive sites (DHSs), termed +55, +58, and +62. Here we utilized a human adult-stage erythroid cell line to show by CRISPR-Cas9 mediated targeted deletion that the composite enhancer is required both for BCL11A expression and HbF repression. Because deletion of the entire enhancer is currently too inefficient to consider for a gene editing approach to hemoglobin disorders, we sought to define the critical features of the enhancer in its natural genomic context. We designed and synthesized a tiling pooled guide RNA (gRNA) library to conduct saturating mutagenesis of the enhancer sequences in situ using the CRISPR-Cas9 gene editing platform. The gRNAs direct Cas9 cleavage and non-homologous end-joining repair at discrete sites throughout the enhancer. By comparing the representation of lentiviral gRNA integrants in high and low HbF pools of the adult erythroid cells, we generated a functional map approaching nucleotide resolution of sequences within the enhancer influencing BCL11A regulation. We observed several discrete enhancer regions required for maximal expression. The largest effect was observed by producing mutations within a narrow functional core of the +58 DHS. These sequences include a GATA1 motif conserved among vertebrates located within a primate-specific context. This region constitutes an Achilles Heel for functional inactivation of the enhancer. We also identified rare genetic variants within the +58 DHS core in individuals with sickle cell disease that are associated with HbF level, independent of all known associations of common genetic variants. In parallel, we performed a similar saturating CRISPR mutagenesis screen of the corresponding murine Bcl11a enhancer. To our surprise, despite low-resolution evidence of conservation by primary sequence homology, syntenic genomic position, and shared chromatin signature, the mouse enhancer sequence determinants of BCL11A expression showed substantial functional divergence. The +58 orthologous sequences were dispensable whereas the +62 orthologous sequences were critically required in murine adult erythroid cells. These results were validated by producing targeted deletions in mouse and human adult erythroid cell lines. Furthermore we subjected cells to individual gRNAs to correlate individual nucleotide disruptions with loss of BCL11A expression. To substantiate the tissue-restricted effect of the enhancer mutations, we generated transgenic mice with deletion of the Bcl11a enhancer and found these sequences were dispensable for expression in developing neurons and B-lymphocytes (unlike conventional Bcl11a knockout) but essential for appropriate hemoglobin switching in vivo. We showed that in primary CD34+ hematopoietic stem and progenitor derived human erythroid precursors that delivery of an individual gRNA and Cas9 is sufficient to produce robust reinduction of HbF. These results validate the BCL11A erythroid enhancer as a promising therapeutic target. Our findings define the most favorable regions for generation of indel mutations in the BCL11A erythroid enhancer as a therapeutic genome editing strategy for HbF reinduction for the β-hemoglobin disorders. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zhang:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Horizon Discovery: Membership on an entity's Board of Directors or advisory committees. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

A 44-year-old male patient with lupus erythematosus profundus (lupus panniculitis) (LEP) of the right cheek/infraorbital region presented as a recurrent swelling. He had only anti-neutrophil-cytoplasmic antibodies (ANCA), which have not been previously described in association with LEP. There was no indication of systemic lupus erythematosus. While chloroquine produced no benefits, the skin lesions promptly cleared with dapsone. Step by step we reduced dapsone to a final dose of 50-75 mg per week. The ANCA titer decreased in parallel to the clinical improvement.

In vivo 1H MRS can be used to detect and quantify the lactate resonance at 1.3 ppm provided that overlapping lipid resonances are eliminated. A homonuclear spectral editing method was developed to acquire uncontaminated 1H spectra of lactate with adiabatic pulses. An advantage of the adiabatic pulse sequence is the ability to induce uniform flip angles and to maximize sensitivity in applications employing surface coil transmitters which produce highly inhomogeneous B1. Glycolytic activity in an intracerebral C6 glioma in rats was monitored by using adiabatic editing sequences to observe [3-13C]lactate produced from infused [1-13C]glucose. Acute hyperglycemia (serum glucose > 22 mM, n = 10) had no significant effect (P = 0.08) on the total ([12C] + [13C]) tumor lactate signal intensity.

The QUEGS facility at MURR has produced a number of new results and demonstrated the range of potential applications of high resolution, high intensity Moessbauer scattering. This work has been carried out by both MU and Purdue researchers and includes published results on Na, W, pentadecane, polydimethylsiloxane and other systems, manuscripts submitted on alkali halides (Phys. Rev. B) and accurate Moessbauer lineshape measurements (Phys. Rev. C), and manuscripts in preparation on glycerol, NiAl and Moessbauer spectra obtained by modulating a scattering crystal. Recently, new collaborations have been initiated which will substantially enhance our efforts. These are with W. Steiner (Vienna), G. Coddens (Saclay), and R. D. Taylor (Los Alamos). Steiner is experienced with Fe-57 Moessbauer scattering, while Coddens specializes in quasielastic neutron scattering; both of these areas naturally complement our work. R. D. Taylor has pioneered Moessbauer spectroscopy from the time of its discovery and has already made important contributions to our study of lattice dynamics and superconductivity for lead alloyed with small quantities of tin. At the same time, a significant instrument upgrade is underway, funded in part by the DOE-URIP program.

We give a progress report for the work which has been carried out in the last three years with DOE support. A facility for high-intensity Moessbauer scattering is now fully operational at the University of Missouri Research Reactor (MURR) as well as facility at Purdue, using special isotopes produced at MURR. High precision, fundamental Moessbauer effect studies have been carried out using scattering to filter the unwanted radiation. These have led to a new Fourier transform method for describing Moessbauer effect (ME) lineshape and a direct method of fitting ME data to the convolution integral. These methods allow complete correction for source resonance self absorption (SRSA) and the accurate representation of interference effects that add an asymmetric component to the ME lines. We have begun applying these techniques to attenuated ME sources whose central peak has been attenuated by stationary resonant absorbers, to more precisely determine interference parameters and line-shape behavior in the resonance asymptotic region. This analysis is important to both the fundamental ME studies and to scattering studies for which a deconvolution is essential for extracting the correct recoilless fractions and interference parameters. A number of scattering studies have been successfully carried out including a study of themore » thermal diffuse scattering in Si, which led to an analysis of the resolution function for gamma-ray scattering. Also studied was the anharmonic motion in Na and the satellite reflection Debye-Waller factor in TaS{sub 2}, which indicate phason rather than phonon behavior. We have begun quasielastic diffusion studies in viscous liquids and current results are summarized. These advances, coupled to our improvements in MIcrofoil Conversion Electron spectroscopy lay the foundation for the proposed research outlined in this request for a three-year renewal of DOE support.« less

A recently developed M\"ossbauer instrument has been used to study the liquid dynamics of pentadecane at various temperatures and scattering angles. Widths of the quasielastic signal extracted from the data ranged between 16 GHz (at 12 \ifmmode^\circ\else\textdegree\fi{}C) and 48 GHz (at 74 \ifmmode^\circ\else\textdegree\fi{}C) at a scattering vector of 1.15 A${\mathrm{\r{}}}^{\mathrm{\ensuremath{-}}1}$. Similar measurements at five different scattering angles (at 12 \ifmmode^\circ\else\textdegree\fi{}C) were also taken to investigate the Q dependence between 0.82 and 1.56 A${\mathrm{\r{}}}^{\mathrm{\ensuremath{-}}1}$. The results obtained are in good agreement with classical theory for coherent scattering from liquids.

The corrections made in a recent paper to the published values for double K-shell ionization in the electron capture decays of $^{54}\mathrm{Mn}$ and $^{65}\mathrm{Zn}$ are not applicable to the data from which these values were derived. Attention is called to a recent article that is relevant to the topic of the paper.

We report a simple analytic form for the convolution integral in transmission Moessbauer spectroscopy allowing accurate representation of the line shape even for very thick absorbers (t = 10), and permitting easy fitting to the true line-shape function. This representation permits the accurate determination of all Moessbauer-effect (ME) parameters, including position, width, cross section, and interference. This analytic method can be applied to deconvolute accurately information contained in either source or absorber, and an explicit analytic form for the emission and absorption Fourier transforms is given. We show that from the asymptotics of the line shape, it is possible to determine all line-shape parameters, and that line-shape asymptotics can circumvent short-ranged hyperfine or instrumental broadening contributions to the observed spectrum. A formula for the correction to the line shape caused by source self-absorption is given, and it is shown that when there is significant source resonance self-absorption a ''good'' fit to data, judged by a chi-squared analysis, can yield completely wrong ME line-shape parameters. We find an equation for the dependence of the area under the absorption curve and the resonance peak height, and give its explicit dependence on the interference parameter and source broadening parameters. Although these effects havemore » been neglected in earlier work, their contribution may be of order 10% in many cases of interest.« less

The total K-ionization probabilities accompanying the ..beta../sup +/ and ..beta../sup -/ decays of /sup 64/Cu, P/sub K/(..beta../sup +/), and P/sub K/(..beta../sup -/) were determined from coincidence and singles K x-ray spectra of Ni and Zn to be (13.2 +- 0.8) x 10/sup -4/ and (11.9 +- 0.8) x 10/sup -4/, respectively, using a high resolution Si(Li) photon spectrometer. P/sub K/(..beta../sup +/)/P/sub K/(..beta../sup -/) was measured directly and independently to be 1.26 +- 0.10, in fair agreement with the ratio of the separate measurements 1.11 +- 0.10. While the average ratio is in agreement with the shaking theory of Isozumi, Shimizu, and Mukoyama, the individual values are a factor of approx. 1.5 larger than their predictions. A similar approximately twofold excess over the Isozumi, Shimizu, and Mukoyama hydrogenic-wave-function-based predictions has been observed for almost all cases of K-shell shaking in ..beta../sup -/ decay, and now also for this first reported measurement in ..beta../sup +/ decay. The measured ratio is the first experimental evidence affirming the previously recognized and acknowledged error of a factor of 2 (excess) for shakeoff in ..beta../sup -/ decay (but not in ..beta../sup +/ decay) in the theory of Law and Campbell. No evidence was found for amore » postulated Ni K vacancy production mechanism, in which the positron annihilates with a K electron internal to the parent atom, exceeding a few percent of K shaking in positron decay.« less

The Debye-Waller factor of sodium has been measured as a function of temperature from 80 to 295 K using Moessbauer ..gamma..-ray scattering. The high energy resolution provided by this technique allowed experimental separation of the elastic scattering from the inelastic thermal diffuse scattering. The results were compared with the harmonic model using integrations over dispersion curves from the neutron-scattering measurements of Woods et al. and the lattice-dynamics calculations of Glyde and Taylor. The Debye-Waller exponent was shown to exceed the harmonic prediction by 23% at room temperature. This difference is attributed to anharmonic terms in the interatomic potential.

A microfoil internal conversion electron (MICE) detector is described which permits direct Doppler shifting of resonance radiation up to velocities of ± 20 cm/s, and gives large improvements in signal-to-background ratios for many Mossbauer isotopes, when compared with transmission geometry. The detector described has efficiency of nearly unity, and it allows for cooling the reciprocating microfoil module to 100 K, which improves signal-to-background substantially over room temperature operation. We give an analysis of the signal-to-background that can be expected for this MICE detector, and for a corresponding transmission experiment. In a table of neutron produced isotopes we find more than 10 cases which are favorable to the MICE approach as compared with the more conventional transmission geometry. The signal-to-background enhancement predicted for several Mossbauer isotopes is substantial for the MICE geometry compared to transmission geometry. Direct measurements of the Mossbauer conversion electron spectrum for the 46.5 and 99.1 keV transitions of 183 W and the 100.1 keV transition in 182 W are reported and compared with our analysis. In the case of 183 W (46.5 keV) we observe over 500% signal-to-background, and this experimental result agrees well with our analysis of the expected size of the effect. Satisfactory agreement is also found for the 99.1 keV 183 W and 100.1 keV 182 W spectra. Based on the analysis given it is possible to determine nuclear resonance cross sections and thereby infer internal conversion coefficients for the resonance transitions. Thus, we are able to determine the internal conversion coefficient for the 46.5 keV transition in 183 W to be α = 13 ± 3.