Measurement of the Spectral Shape of the ß-Decay of

We report on a comparison between the theoretically predicted and experimentally measured spectra of the first-forbidden nonunique ß-decay transition ^{137}Xe(7/2^{-})→^{137}Cs(7/2^{+}). The experimental data were acquired by the EXO-200 experiment during a deployment of an AmBe neutron source. The ultralow background environment of EXO-200, together with dedicated source deployment and analysis procedures, allowed for collection of a pure sample of the decays, with an estimated signal to background ratio of more than 99 to 1 in the energy range from 1075 to 4175 keV. In addition to providing a rare and accurate measurement of the first-forbidden nonunique ß-decay shape, this work constitutes a novel test of the calculated electron spectral shapes in the context of the reactor antineutrino anomaly and spectral bump.

Search for Neutrinoless Double-ß Decay with the Complete EXO-200 Dataset.

A search for neutrinoless double-ß decay (0νßß) in ^{136}Xe is performed with the full EXO-200 dataset using a deep neural network to discriminate between 0νßß and background events. Relative to previous analyses, the signal detection efficiency has been raised from 80.8% to 96.4±3.0%, and the energy resolution of the detector at the Q value of ^{136}Xe 0νßß has been improved from σ/E=1.23% to 1.15±0.02% with the upgraded detector. Accounting for the new data, the median 90% confidence level 0νßß half-life sensitivity for this analysis is 5.0×10^{25} yr with a total ^{136}Xe exposure of 234.1 kg yr. No statistically significant evidence for 0νßß is observed, leading to a lower limit on the 0νßß half-life of 3.5×10^{25} yr at the 90% confidence level.

Genetic programming of macrophages to perform anti-tumor functions using targeted mRNA nanocarriers.

Tumor-associated macrophages (TAMs) usually express an M2 phenotype, which enables them to perform immunosuppressive and tumor-promoting functions. Reprogramming these TAMs toward an M1 phenotype could thwart their pro-cancer activities and unleash anti-tumor immunity, but efforts to accomplish this are nonspecific and elicit systemic inflammation. Here we describe a targeted nanocarrier that can deliver in vitro-transcribed mRNA encoding M1-polarizing transcription factors to reprogram TAMs without causing systemic toxicity. We demonstrate in models of ovarian cancer, melanoma, and glioblastoma that infusions of nanoparticles formulated with mRNAs encoding interferon regulatory factor 5 in combination with its activating kinase IKKß reverse the immunosuppressive, tumor-supporting state of TAMs and reprogram them to a phenotype that induces anti-tumor immunity and promotes tumor regression. We further establish that these nanoreagents are safe for repeated dosing. Implemented in the clinic, this immunotherapy could enable physicians to obviate suppressive tumors while avoiding systemic treatments that disrupt immune homeostasis.

Search for Neutrinoless Double-Beta Decay with the Upgraded EXO-200 Detector.

Results from a search for neutrinoless double-beta decay (0νßß) of ^{136}Xe are presented using the first year of data taken with the upgraded EXO-200 detector. Relative to previous searches by EXO-200, the energy resolution of the detector has been improved to σ/E=1.23%, the electric field in the drift region has been raised by 50%, and a system to suppress radon in the volume between the cryostat and lead shielding has been implemented. In addition, analysis techniques that improve topological discrimination between 0νßß and background events have been developed. Incorporating these hardware and analysis improvements, the median 90% confidence level 0νßß half-life sensitivity after combining with the full data set acquired before the upgrade has increased twofold to 3.7×10^{25} yr. No statistically significant evidence for 0νßß is observed, leading to a lower limit on the 0νßß half-life of 1.8×10^{25} yr at the 90% confidence level.

Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers.

Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knock-out selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.Current widely used viral and electroporation methods for creating therapeutic cell-based products are complex and expensive. Here, the authors develop targeted mRNA nanocarriers that can transiently program gene expression by simply mixing them with cells, to improve their therapeutic potential.

An apparatus to manipulate and identify individual Ba ions from bulk liquid Xe.

We describe a system to transport and identify barium ions produced in liquid xenon, as part of R&D towards the second phase of a double beta decay experiment, nEXO. The goal is to identify the Ba ion resulting from an extremely rare nuclear decay of the isotope (136)Xe, hence providing a confirmation of the occurrence of the decay. This is achieved through Resonance Ionization Spectroscopy (RIS). In the test setup described here, Ba ions can be produced in liquid xenon or vacuum and collected on a clean substrate. This substrate is then removed to an analysis chamber under vacuum, where laser-induced thermal desorption and RIS are used with time-of-flight mass spectroscopy for positive identification of the barium decay product.

New Diversity Arrays Technology (DArT) markers for tetraploid oat (Avena magna Murphy et Terrell) provide the first complete oat linkage map and markers linked to domestication genes from hexaploid A. sativa L.

Nutritional benefits of cultivated oat (Avena sativa L., 2n = 6x = 42, AACCDD) are well recognized; however, seed protein levels are modest and resources for genetic improvement are scarce. The wild tetraploid, A. magna Murphy et Terrell (syn A. maroccana Gdgr., 2n = 4x = 28, CCDD), which contains approximately 31% seed protein, was hybridized with cultivated oat to produce a domesticated A. magna. Wild and cultivated accessions were crossed to generate a recombinant inbred line (RIL) population. Although these materials could be used to develop domesticated, high-protein oat, mapping and quantitative trait loci introgression is hindered by a near absence of genetic markers. Objectives of this study were to develop high-throughput, A. magna-specific markers; generate a genetic linkage map based on the A. magna RIL population; and map genes controlling oat domestication. A Diversity Arrays Technology (DArT) array derived from 10 A. magna genotypes was used to generate 2,688 genome-specific probes. These, with 12,672 additional oat clones, produced 2,349 polymorphic markers, including 498 (21.2%) from A. magna arrays and 1,851 (78.8%) from other Avena libraries. Linkage analysis included 974 DArT markers, 26 microsatellites, 13 SNPs, and 4 phenotypic markers, and resulted in a 14-linkage-group map. Marker-to-marker correlation coefficient analysis allowed classification of shared markers as unique or redundant, and putative linkage-group-to-genome anchoring. Results of this study provide for the first time a collection of high-throughput tetraploid oat markers and a comprehensive map of the genome, providing insights to the genome ancestry of oat and affording a resource for study of oat domestication, gene transfer, and comparative genomics.

EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding.

The EPR spectra of NH(2)-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g approximately 8, 4, 2). A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L. A rhombic high spin signal (g approximately 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome. Accompanying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g approximately 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4.

Promoter function and the role of cytokines in the transcriptional regulation of rabbit CYP2E1 and CYP2E2.

Rabbit CYP2E1 and CYP2E2 show considerable similarity in the 5' flanking region, but a 32-base-pair element (32-BPE) that is repeated in 2E1 is present only as a single inexact copy in 2E2. In the present investigation, footprinting disclosed two specific binding sites for liver nuclear proteins, and the DNase I sensitivity profiles of the two genes were found to be different. Several positive and negative regulatory elements were identified by transfection with a series of constructs of upstream CYP2E sequences fused to the luciferase gene. Both genes have an HNF-1 consensus motif with one nucleotide mismatch, which affects binding affinity and promoter activity. Investigation of DNA-protein interactions revealed that Sp1 and NFkappaB bind exclusively to the 32-BPE of 2E1 and 2E2, respectively, suggesting a possible regulatory role for the 32-BPE. Interleukin-1alpha (IL-1alpha) gave rise to a 2.5-fold increase in the promoter activity of 2E1 in HepG2 cells, and the IL-1alpha-mediated induction of reporter gene expression was almost completely prevented when the 32-BPE was deleted. Increased DNA binding and Sp1 protein content as a result of IL-1alpha treatment, as well as cotransfection experiments with pPacSp1, suggest that Sp1 is a transcription activator for the induction of 2E1 by IL-1alpha in HepG2 cells.

Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes.

The reaction of hydrogen peroxide and certain aromatic aldehydes with cytochrome P450BM3-F87G results in the covalent modification of the heme cofactor of this monooxygenase. Analysis of the resulting heme by electronic absorption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P450(2B4), which apparently occurs via a peroxyhemiacetal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was observed that replacement of the Phe-87 in the P450BM3 by the smaller glycyl residue was essential for the modification to proceed, as the wild-type enzyme showed no spectral changes under identical conditions. The kinetics of this reaction were examined by stopped-flow spectrophotometry with 3-phenylpropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the process of heme modification was biphasic, with initial bleaching of the Soret absorbance, followed by an increase in absorbance centered at 430 nm, consistent with meso-heme adduct formation. The intermediate formed during phase I also showed an increased absorbance between 700 and 900 nm, relative to the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for phase II. All of these observations are consistent with a mechanism involving radical attack at the gamma-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of which produces the gamma-meso-alkyl heme derivative.

Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E.

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.

Selective pharmacologic inhibition of murine and human IL-12-dependent Th1 differentiation and IL-12 signaling.

We have previously shown that lisofylline (LSF) inhibits murine Th1-mediated disease in vivo by blocking IL-12-induced differentiation of Th1 cells. The cellular and molecular mechanisms underlying this inhibition were further explored by testing LSF in several IL-12-responsive model systems in vitro. IL-12-dependent Th1 differentiation was abrogated by LSF and yielded effector T cells that were deficient in proinflammatory cytokine secretion, including IFN-gamma, IL-2, and TNF-alpha. The diminished Th1 phenotype resulted from both a lower frequency of IL-12-derived Th1 clones and a reduced capacity of individual clones to secrete IFN-gamma due to lower levels of IFN-gamma mRNA. The arrest in Th1 development resulted from a blockade of IL-12 signaling that preceded the Th0 to Th1 transition. Thus, LSF blocked IL-12-enhanced IFN-gamma production in anti-CD3-stimulated T cells and prevented IL-12-mediated repression of the transcription factor GATA-3. Lisofylline also inhibited IL-12-induced increases in STAT4 tyrosine phosphorylation, but did not block TCR signaling or inhibit acquisition of IL-12 responsiveness. These findings were extended to show that LSF also inhibits IL-12-dependent responses in human T cells. LSF, which has one asymmetric chiral center, was selectively inhibitory for IL-12 signaling compared with its S-enantiomer (1501-S) and the oxidized side chain analog, pentoxifylline. The results suggest that LSF may be useful as a modulator of Th1-mediated disease in humans.

Expression of MXI1, a Myc antagonist, is regulated by Sp1 and AP2.

MXI1, a member of the MAD family of Myc antagonists, encodes a transcription factor whose expression must be tightly regulated to maintain normal cell growth and differentiation. To more closely investigate the transcriptional regulation of the human MXI1 gene, we have cloned and characterized the MXI1 promoter. After clarification of the 5'- and 3'-untranslated regions of the cDNA (indicating that the true length of the MXI1 transcript is 2643 base pairs), we identified two transcription initiation sites. We subsequently isolated the MXI1 promoter, which is GC-rich and lacks a TATA box. Although it contains at least six potential initiator sequences, functional studies indicate the proximal two initiator sequences in combination with nearby Sp1 and MED-1 sites together account for virtually all promoter activity. We also demonstrate that MXI1 promoter activity is repressed by high levels of AP2. These studies provide further insight into the complex regulatory mechanisms governing MXI1 gene expression and its role in cellular differentiation and tumor suppression.

Discrete species of activated oxygen yield different cytochrome P450 heme adducts from aldehydes.

Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH-cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W. , Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O(2)-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy-iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the gamma-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen-oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes.

Prevention of experimental allergic encephalomyelitis via inhibition of IL-12 signaling and IL-12-mediated Th1 differentiation: an effect of the novel anti-inflammatory drug lisofylline.

Experimental allergic encephalomyelitis (EAE) is an inflammatory, CD4+ Th1-mediated autoimmune disease, which serves as a model for multiple sclerosis. We examined the effect of a novel anti-inflammatory drug, lisofylline (LSF), on EAE induced either by injection of mouse spinal cord homogenate or following transfer of myelin basic protein-reactive T cells. Orally administered LSF significantly inhibited EAE in both cases, decreasing peak clinical scores by >70% and >80%, respectively. In addition, analysis of representative spinal cord sections from LSF-treated mice showed complete lack of demyelination and lymphocyte infiltration. The reduction in EAE correlated with the inhibition of Th1 differentiation by LSF in vivo, as indicated by a reduction in T cell IFN-gamma production ex vivo after Ag restimulation. The inhibition of Th1 differentiation in vivo is consistent with a block in IL-12 receptor signaling, because LSF blocked IL-12-driven Th1 differentiation and T cell proliferation in vitro, yet had no effect on IL-12 secretion from APCs ex vivo or in vitro.

Multiple activated oxygen species in P450 catalysis: contributions To specificity in drug metabolism.

A hypervalent iron-oxene species has been widely proposed as the "active oxygen" in cytochrome P450 (P450)-catalyzed reactions. We recently examined the effect of mutation of the highly conserved threonine residue in P450s 2B4 and 2E1 to alanine, a change that is believed to interfere with proton delivery to the active site, and have determined the change in rates of deformylation of aldehydes, epoxidation of olefins, and hydroxylation of various substrates. The results support the concept that three distinct oxidants are functional in P450 catalysis: nucleophilic peroxo-iron, nucleophilic or electrophilic hydroperoxo-iron, and electrophilic oxenoid-iron. The occurrence of multiple oxidizing species may contribute to the remarkable versatility of the P450 family of isozymes in the modification of drugs and other substrates. Furthermore, the relative concentrations of these oxidants in a particular P450 isozyme may contribute to substrate specificity and govern the type of reaction catalyzed.

Membrane topology of cytochrome P450 2B4 in Langmuir-Blodgett monolayers.

Using Langmuir-Blodgett monolayers of both phosphatidylethanolamines and phosphatidylcholines as membrane mimics, we have examined the topology of cytochrome P450 2B4 anchoring. The interaction of wild-type P450 2B4 with phosphatidylethanolamine monolayers can be characterized as a biphasic reaction, with the initial fast phase explained by the specific insertion of membrane-spanning segments of the protein into the monolayer. Injection of cytochrome b5 (b5) beneath dipalmitoyl-phosphatidylcholine monolayers also resulted in biphasic kinetics. Regardless of the nature of the lipid employed, neither a truncated cytochrome P450 2B4 (P450 2B4 Delta2-27) lacking the amino-terminal hydrophobic residues widely believed to be the major transmembrane segment nor a soluble b5 fragment (Deltab5) lacking its carboxy terminus anchor exhibit the fast-phase behavior characteristic of specific insertion. To further characterize the membrane topology of P450 2B4, its insertion area in DPPE monolayers was measured and analyzed with use of the Gibbs equation for adsorption at an interface. The mean molecular insertion area derived from isotherms of P450 2B4 in a DPPE monolayer at a pressure of 19 mN/m, 680 +/- 95 A2 is large enough to accommodate two to four transmembrane helices. The large insertion area and the fact that the truncated cytochrome retains as much as 30% of its membrane localization when expressed in Escherichia coli (Pernecky, S. J., Larson, J. R., Philpot, R. M., and Coon, M. J. (1993) Proc. Natl. Acad. Sci. USA 90, 2651-2655) suggest that this cytochrome is not deeply embedded but that other regions, in addition to the amino-terminal 26 residues, may be involved in the interaction of cytochrome P450 with the membrane.

Cytochrome P450 2E1 mRNA in the rat prostate: detection and quantitation by competitive reverse transcription and polymerase chain reaction.

Cytochrome P450 2E1 plays a pivotal role in the metabolic activation of a wide variety of low molecular weight environmental toxicants and procarcinogens. In the present study, expression of the P450 2E1 gene in the rat prostate gland was quantitated by competitive reverse transcription and the polymerase chain reaction. To assess accurately the induction level of P450 2E1 mRNA in the prostate after pyridine treatment of rats, a recombinant standard RNA was generated that is homologous to the sequence of P450 2E1 mRNA except for an internal deletion of 100 bases. The data indicate that P450 2E1 mRNA is present in the prostate of untreated animals and is induced about four-fold by treatment with pyridine. The results suggest that exposure to certain environmental chemicals and procarcinogens may increase P450 2E1 levels in the prostate gland and thus could enhance formation of reactive, carcinogenic metabolites.

Regulation of rabbit cytochrome P450 2E1 expression in HepG2 cells by insulin and thyroid hormone.

The regulation of cytochrome P450 (CYP) 2E1, the ethanol-inducible isoform, is particularly complex. The level is affected by a variety of other foreign compounds, by insulin (as studied in several laboratories), and by triiodothyronine (T3), which has not been previously examined at the molecular level. In the present investigation, a stably transfected HepG2 cell line harboring a rabbit CYP2E1 minigene containing the coding sequence together with 1.6 kilobases of the 5' flanking region and the untranslated region (UTR), as well as 0.5 kilobases of the 3' UTR, was established. Western blot analysis showed that 1 microM insulin decreased the CYP2E1 protein level in a dose- and time-dependent manner, whereas 1 microM T3 increased the level 2-fold in 1 day and 8-fold in 5 days. Similarly, steady state CYP2E1 mRNA levels were decreased by insulin but were increased by T3. Neither hormone affected the transcription rate of the CYP2E1 5' flanking region with an UTR/luciferase fusion gene, indicating that the regulation is post-transcriptional in this system under our experimental conditions. When the CYP2E1 3' UTR was removed from the minigene, CYP2E1 mRNA and protein were up-regulated by insulin but were not affected by T3. These findings, including mRNA half-life determinations, indicate that the effects of insulin and T3 are a result of altered mRNA stability and that the 3' UTR of CYP2E1 contains regulatory information for these hormone-mediated effects.