Confidence limits in pulse dipolar EPR spectroscopy: estimates for individual measurements.

The distribution of inter-label distances obtained by electron paramagnetic resonance (EPR) pulse dipolar spectroscopies (PDS), such as DEER aka PELDOR, gives a valuable characterization of structure on the nanometer scale. The impact of random experimental noise on such experiments is examined for three independent methods for analysing PDS data: the model-free method with Tikhonov regularization, model-free with Mellin-transformation, and a model-based method. All three methods show negative bias for the mean distance and positive bias for the distribution width. Both biases grow with increasing noise levels. The estimated confidence bands and the uncertainties obtained from a single experimental measurement by the standard bootstrapping or χ2-surface scanning approaches are inconsistent and can exclude the true distance distribution. Yet, both approaches can provide quite valuable support for hypothesis testing in PDS studies.

Forming a chromium-based interstrand DNA crosslink: Implications for carcinogenicity.

The reduction of the carcinogen chromate has been proposed to lead to three Cr(III)-containing DNA lesions: binary adducts (Cr(III) and DNA), interstrand crosslinks, and ternary adducts (Cr(III) linking DNA to a small molecule or protein). Although the structures of binary adducts have recently been elucidated, the structures of interstrand crosslinks and ternary adducts are not known. Analysis of Cr(III) binding to an oligonucleotide duplex containing a 5'-CG site allows elucidation of the structure of an oxide- or hydroxide-bridged binuclear Cr(III) assembly bridging the two strands of DNA. One Cr(III) is directly coordinated by the N-7 atom of a guanine residue, and the complex straddles the helix to form a hydrogen bond between another guanine residue and a Cr(III)-bound aquo ligand. No involvement of the phosphate backbone was observed. The properties and stability of this Cr-O(H)-Cr-bridged complex differ significantly from those reported for Cr-induced interstrand crosslinks, suggesting that interstrand crosslinks resulting from chromate reduction may be organic in nature.

Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium.

The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.

Divergent Metabolic Changes in Rhizomes of Lowland and Upland Switchgrass (Panicum virgatum) from Early Season through Dormancy Onset.

High-biomass-yielding southerly adapted switchgrasses (Panicum virgatum L.) frequently suffer from unpredictable winter hardiness at more northerly sites arising from damage to rhizomes that prevent effective spring regrowth. Previously, changes occurring over the growing season in rhizomes sampled from a cold-adapted tetraploid upland cultivar, Summer, demonstrated a role for abscisic acid (ABA), starch accumulation, and transcriptional reprogramming as drivers of dormancy onset and potential keys to rhizome health during winter dormancy. Here, rhizome metabolism of a high-yielding southerly adapted tetraploid switchgrass cultivar, Kanlow-which is a significant source of genetics for yield improvement-was studied over a growing season at a northern site. Metabolite levels and transcript abundances were combined to develop physiological profiles accompanying greening through the onset of dormancy in Kanlow rhizomes. Next, comparisons of the data to rhizome metabolism occurring in the adapted upland cultivar Summer were performed. These data revealed both similarities as well as numerous differences in rhizome metabolism that were indicative of physiological adaptations unique to each cultivar. Similarities included elevated ABA levels and accumulation of starch in rhizomes during dormancy onset. Notable differences were observed in the accumulation of specific metabolites, the expression of genes encoding transcription factors, and several enzymes linked to primary metabolism.

Sugars and cuticular waxes impact sugarcane aphid (Melanaphis sacchari) colonization on different developmental stages of sorghum.

Sugarcane aphid (SCA; Melanaphis sacchari) is a devastating pest of sorghum (Sorghum bicolor) that colonizes sorghum plants at different growth stages. Leaf surface characteristics and sugars often influence aphid settling and feeding on host plants. However, how changes in cuticular waxes and sugar levels affect SCA establishment and feeding at different development stages of sorghum have not been explored. In this study, two- and six-week-old BTx623 plants, a reference line of sorghum, was used to evaluate plant-aphid interactions. Monitoring aphid feeding behavior using Electrical Penetration Graph (EPG) technique revealed that aphids spent more time in the sieve element phase of six-week-old plants compared to two-week-old plants. Significant differences were found in the time spent to reach the first sieve element and pathway phases between the two- and six-week-old plants. However, no-choice aphid bioassays displayed that SCA population numbers were higher in two-week-old plants compared to six-week-old plants. Differences in the abundance of wax and sugar contents were analyzed to determine how these plant components influenced aphid feeding and proliferation. Among the cuticular wax compounds analyzed, α-amyrin and isoarborinone increased after 10 days of aphid infestation only in six-week-old plants. Trehalose content was significantly increased by SCA feeding on two- and six-week-old plants. Furthermore, SCA feeding depressed sucrose content and increased levels of glucose and fructose in two-week-old but not in six-week-old plants. Overall, our study indicates that plant age is a determinant for SCA feeding, and subtle changes in triterpenoids and available sugars influence SCA establishment on sorghum plants.

Autoinhibitory structure of preligand association state implicates a new strategy to attain effective DR5 receptor activation.

Members of the tumor necrosis factor receptor superfamily (TNFRSF) are important therapeutic targets that can be activated to induce death of cancer cells or stimulate proliferation of immune cells. Although it has long been implicated that these receptors assemble preligand associated states that are required for dominant interference in human disease, such states have so far eluded structural characterization. Here, we find that the ectodomain of death receptor 5 (DR5-ECD), a representative member of TNFRSF, can specifically self-associate when anchored to lipid bilayer, and we report this self-association structure determined by nuclear magnetic resonance (NMR). Unexpectedly, two non-overlapping interaction interfaces are identified that could propagate to higher-order clusters. Structure-guided mutagenesis indicates that the observed preligand association structure is represented on DR5-expressing cells. The DR5 preligand association serves an autoinhibitory role as single-domain antibodies (sdAbs) that partially dissociate the preligand cluster can sensitize the receptor to its ligand TRAIL and even induce substantial receptor signaling in the absence of TRAIL. Unlike most agonistic antibodies that require multivalent binding to aggregate receptors for activation, these agonistic sdAbs are monovalent and act specifically on an oligomeric, autoinhibitory configuration of the receptor. Our data indicate that receptors such as DR5 can form structurally defined preclusters incompatible with signaling and that true agonists should disrupt the preligand cluster while converting it to signaling-productive cluster. This mechanism enhances our understanding of a long-standing question in TNFRSF signaling and suggests a new opportunity for developing agonistic molecules by targeting receptor preligand clustering.

Experimental Observation of a Peculiar Effect in Saturated Electron Paramagnetic Resonance Spectra Undergoing Spin Exchange. Magnetic Polariton?

We report the experimental observation of a spectral manifestation of a magnetic polariton that was theoretically predicted last year. This unprecedented manifestation is demonstrated not only for 15N-enriched peroxylamine disulfonate, a radical that adheres strictly to the assumptions of the theory, but also for a radical, 4-oxo-2,2,6,6-tetramethylpiperidine-d16;1-15N-1-oxyl, that departs somewhat from the assumptions, as well as the Galvinoxyl radical that represents a severe departure. The magnetic polariton is likely to be of interest to physical chemists in other fields because of the intrinsic advantage of a finite basis set in developing theories.

Microbial dysbiosis and childhood asthma development: Integrated role of the airway and gut microbiome, environmental exposures, and host metabolic and immune response.

Asthma is a chronic and heterogeneous respiratory disease with many risk factors that typically originate during early childhood. A complex interplay between environmental factors and genetic predisposition is considered to shape the lung and gut microbiome in early life. The growing literature has identified that changes in the relative abundance of microbes (microbial dysbiosis) and reduced microbial diversity, as triggers of the airway-gut axis crosstalk dysregulation, are associated with asthma development. There are several mechanisms underlying microbial dysbiosis to childhood asthma development pathways. For example, a bacterial infection in the airway of infants can lead to the activation and/or dysregulation of inflammatory pathways that contribute to bronchoconstriction and bronchial hyperresponsiveness. In addition, gut microbial dysbiosis in infancy can affect immune development and differentiation, resulting in a suboptimal balance between innate and adaptive immunity. This evolving dysregulation of secretion of pro-inflammatory mediators has been associated with persistent airway inflammation and subsequent asthma development. In this review, we examine current evidence around associations between the airway and gut microbial dysbiosis with childhood asthma development. More specifically, this review focuses on discussing the integrated roles of environmental exposures, host metabolic and immune responses, airway and gut microbial dysbiosis in driving childhood asthma development.

Magnetic Ordering in a High-Spin Donor-Acceptor Conjugated Polymer.

The development of open-shell organic molecules that magnetically order at room temperature,which can be practically applied, remains a grand challenge in chemistry, physics, and materials science. Despite the exploration of vast chemical space, design paradigms for organic paramagnetic centers generally result in unpaired electron spins that are unstable or isotropic. Here, a high-spin conjugated polymer is demonstrated, which is composed of alternating cyclopentadithiophene and benzo[1,2-c;4,5-c']bis[1,2,5]thiadiazole heterocycles, in which macromolecular structure and topology coalesce to promote the spin center generation and intermolecular exchange coupling. Electron paramagnetic resonance (EPR) spectroscopy is consistent with spatially localized spins, while magnetic susceptibility measurements show clear anisotropic spin ordering and exchange interactions that persist at room temperature. The application of long-range π-correlations for spin center generation promotes remarkable stability. This work offers a fundamentally new approach to the implementation of this long-sought-after physical phenomenon within organic materials and the integration of manifold properties within emerging technologies.

Fusarium head blight resistance exacerbates nutritional loss of wheat grain at elevated CO2.

The nutritional integrity of wheat is jeopardized by rapidly rising atmospheric carbon dioxide (CO2) and the associated emergence and enhanced virulence of plant pathogens. To evaluate how disease resistance traits may impact wheat climate resilience, 15 wheat cultivars with varying levels of resistance to Fusarium Head Blight (FHB) were grown at ambient and elevated CO2. Although all wheat cultivars had increased yield when grown at elevated CO2, the nutritional contents of FHB moderately resistant (MR) cultivars were impacted more than susceptible cultivars. At elevated CO2, the MR cultivars had more significant differences in plant growth, grain protein, starch, fructan, and macro and micro-nutrient content compared with susceptible wheat. Furthermore, changes in protein, starch, phosphorus, and magnesium content were correlated with the cultivar FHB resistance rating, with more FHB resistant cultivars having greater changes in nutrient content. This is the first report of a correlation between the degree of plant pathogen resistance and grain nutritional content loss in response to elevated CO2. Our results demonstrate the importance of identifying wheat cultivars that can maintain nutritional integrity and FHB resistance in future atmospheric CO2 conditions.

Non-uniform sampling in pulse dipolar spectroscopy by EPR: the redistribution of noise and the optimization of data acquisition.

Pulse dipolar spectroscopy (PDS) in Electron Paramagnetic Resonance (EPR) is the method of choice for determining the distance distribution function for mono-, bi- or multi- spin-labeled macromolecules and nanostructures. PDS acquisition schemes conventionally use uniform sampling of the dipolar trace, but non-uniform sampling (NUS) schemes can decrease the total measurement time or increase the accuracy of the resulting distance distributions. NUS requires optimization of the data acquisition scheme, as well as changes in data processing algorithms to accommodate the non-uniformly sampled data. We investigate in silico the applicability of the NUS approach in PDS, considering its effect on random, truncation and sampling noise in the experimental data. Each type of noise in the time-domain data propagates differently and non-uniformly into the distance spectrum as errors in the distance distribution. NUS schemes seem to be a valid approach for increasing sensitivity and/or throughput in PDS by decreasing and redistributing noise in the distance spectrum so that it has less impact on the distance spectrum.

Energetics and kinetics of various cyano radical hydrogen abstractions.

The cyano radical (CN) is an abundant, open-shell molecule found in a variety of environments, including the atmosphere, the interstellar medium and combustion processes. In these environments, it often reacts with small, closed-shell molecules via hydrogen abstraction. Both carbon and nitrogen atoms of the cyano radical are reactive sites, however the carbon is more reactive with reaction barrier heights generally between 2-15 kcal mol-1 lower than those of the analogous nitrogen. The CN + HX → HCN/HNC + X, with X = H, CH3, NH2, OH, F, SiH3, PH2, SH, Cl, C2H, CN reactions have been studied at a high-level of theory, including CCSD(T)-F12a. Furthermore, kinetics were obtained over the 100-1000 K temperature range, showing excellent agreement with those rate constants that have been determined experimentally.

Single-cell mutation analysis of clonal evolution in myeloid malignancies.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Dibridged, Monobridged, Vinylidene-Like, and Linear Structures for the Alkaline Earth Dihydrides Be2H2, Mg2H2, Ca2H2, Sr2H2, and Ba2H2. Proposals for Observations.

This research reports a search for peculiar monobridged structures of the E2H2 molecules (E = Be, Mg, Ca, Sr, Ba). For Be2H2 and Mg2H2, the monobridged geometry is not an equilibrium but rather a transition state between the vinylidene-like structure and the global minimum HE-EH linear geometry. However, for Ca2H2, Sr2H2, and Ba2H2, this situation changes significantly; the linear structure is no longer the global minimum but lies higher in energy than two other equilibria, the dibridged and monobridged structures. The planar dibridged structures of both Sr2H2 and Ba2H2 should be observable via IR spectroscopy. Although the remarkable monobridged structures lie 8.3 (Sr) and 7.6 kcal/mol (Ba) higher, the large IR intensities of the terminal E-H stretching frequencies may make the monobridged structures observable. The monobridged structures have sizable permanent dipole moments (3.07 and 3.06 D for Sr and Ba, respectively) and also should be observable via microwave spectroscopy.

Electron Spin Relaxation of Photoexcited Porphyrin in Water-Glycerol Glass.

Recently, the photoexcited triplet state of porphyrin was proposed as a promising spin-label for pulsed dipolar electron paramagnetic resonance (EPR). Herein, we report the factors that determine the electron spin echo dephasing of the photoexcited porphyrin in a water-glycerol matrix. The electron spin relaxation of a water-soluble porphyrin was measured by Q-band EPR, and the temperature dependence and the effect of solvent deuteration on the relaxation times were studied. The phase memory relaxation rate (1/Tm) is noticeably affected by solvent nuclei and is substantially faster in protonated solvents than in deuterated solvents. The Tm is as large as 13-17 µs in deuterated solvent, potentially expanding the range of distances available for measurement by dipole spectroscopy with photoexcited porphyrin. The 1/Tm depends linearly on the degree of solvent deuteration and can be used to probe the environment of a porphyrin in or near a biopolymer, including the solvent accessibility of porphyrins used in photodynamic therapy. We characterized the noncovalent binding of porphyrin to human serum albumin (HSA) from 1/Tm and electron spin echo envelope modulation (ESEEM) and found that porphyrin is quite exposed to solvent on the surface of HSA. The 1/Tm and ESEEM are equally effective and provide complementary methods to determine the solvent accessibility of a porphyrin bound to protein or to determine the location of the porphyrin.

Changes in Wheat Nutritional Content at Elevated [CO2] Alter Fusarium graminearum Growth and Mycotoxin Production on Grain.

Rising atmospheric [CO2] has been shown to impact plant primary metabolism and the severity of Fusarium head blight (FHB) in wheat. In this study, we evaluated how changes in grain nutritional content due to growth at elevated [CO2] affected Fusarium graminearum growth and mycotoxin production. Susceptible (Norm) and moderately resistant (Alsen) hard spring wheat grains that had been grown at ambient (400 ppm) or elevated [CO2] (800 ppm) were independently inoculated with two F. graminearum fungal strains, which produce the trichothecene mycotoxin, deoxynivalenol. Under higher [CO2], FHB-susceptible and moderately resistant wheat had disproportionate losses in protein and mineral contents, with Alsen being more severely impacted. Furthermore, the F. graminearum strain 9F1 had increased mycotoxin biosynthesis in response to the loss of wheat nutritional content in Alsen. Our results demonstrate that future [CO2] conditions may provide a strain-specific pathogenic advantage on hosts, with greater losses in nutritional content.

Human Serum Albumin Labelled with Sterically-Hindered Nitroxides as Potential MRI Contrast Agents.

Four albumin-nitroxide conjugates were prepared and tested as metal-free organic radical contrast agents (ORCAs) for magnetic resonance imaging (MRI). Each human serum albumin (HSA) carrier bears multiple nitroxides conjugated via homocysteine thiolactones. These molecular conjugates retain important physical and biological properties of their HSA component, and the resistance of their nitroxide groups to bioreduction was retained or enhanced. The relaxivities are similar for these four conjugates and are much greater than those of their individual components: the HSA or the small nitroxide molecules. This new family of conjugates has excellent prospects for optimization as ORCAs.