Radical S-adenosyl-l-methionine (SAM) enzymes figure prominently in the formation of ribosomally synthesized and posttranslationally modified peptides (RiPPs), where they catalyze peptide modifications including epimerization, thioether crosslink formation, and peptide backbone splicing. Here, we use rapid freeze-quench trapping together with electron paramagnetic resonance and electron-nuclear double resonance techniques to probe the mechanistic steps of the two epimerization reactions catalyzed by the radical SAM enzyme EpeE during conversion of its peptide substrate to the epipeptide natural product. Use of the EpeE C223S variant facilitated trapping and characterization of Cα radical intermediates, supporting a central role for C223 in the proposed epimerization mechanism. We showed that both wild-type and C223S EpeE with bound SAM and peptide substrate form the organometallic intermediate Ω upon reaction, and that thermal annealing of Ω results in conversion to an organic radical intermediate. Freeze-quenching at longer times allowed us to directly trap the organic radical intermediate, and isotopic labeling together with use of substrate variants allowed for detailed characterization of the substrate radical intermediates. The results revealed that while LC-MS enzymatic assays point to Ile12 as the initial site of epimerization, freeze-quench EPR reveals that Val4 is the preferred site for initial Cα radical formation. These apparently conflicting results were resolved by the observation that the Ile12 Cα radical is more efficiently quenched to form the d-Ile, thus providing insights into the determinants for substrate binding and epimerization by EpeE.

The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H2. A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that the Escherichia coli iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H2 production rates comparable to that of the in vivo-matured Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [FeII(cys)(CN)(CO)2]- synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (MbH64L) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited Hox-CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and MbH64L to the fully defined maturation system thus results in an in vitro [FeFe]-hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.

Hexaploid wheat has a large genome, making it difficult for transgenes to produce phenotypes due to gene redundancy and tight linkage among genes. Multiple gene copies typically necessitate multiple targeting events during gene editing, followed by several generations of self-crossing to achieve homozygous genotypes. The high cost of transgenesis in wheat is another issue, which hinders the easy availability of gene-edited materials in wheat. In this study, we developed a comprehensive approach to improve wheat gene editing efficiency. First, we established a protoplast-based system to evaluate the relative efficiency of gene editing targets, which enabled the rapid and effective selection of optimal sgRNAs. We then compared two transformation strategies: biolistic bombardment and Agrobacterium-mediated transformation for generating edited wheat lines. Although biolistic bombardment showed higher initial editing efficiency, Agrobacterium-mediated transformation proved more effective for obtaining homozygous mutants. Notably, we discovered that deploying the same sgRNA through different vectors enhanced editing efficiency, whereas overlapping but distinct sgRNAs exhibited interference effects. Finally, we optimized the VITF-edit (virus-induced transgene free editing) technique using BSMV delivery to establish a relatively simple and easily applied wheat gene editing method for general laboratories.

DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.

In this paper, we study the weak irreducibility of stochastic delay differential equations(SDDEs) driven by pure jump noise. The main contribution of this paper is to provide a concise proof of weak irreducibility, releasing condition (A1-3) in Assumption 2.1 from the literature . As an application, we derive the weak irreducibility of SDDEs with weakly dissipative coefficients. An important novelty of this paper is to allow the driving noises to be degenerate. This closes the gap of the irreducibility of SDDEs driven by pure jump noise.

This study presents an experimental evaluation of the Dynamic Extremity SPECT (DE-SPECT) system, specifically engineered for precise, regionselective gamma-ray spectroscopy in the diagnosis of Peripheral Vascular Disease (PVD) in lower extremities. The system incorporates Cadmium Zinc Telluride (CZT) imaging spectrometers and dynamic dual-field-of-view (FOV) collimators to facilitate comprehensive, multifunctional molecular imaging. The CZT detectors, with Depth of Interaction (DOI) capabilities, deliver an exceptional energy performance across a wide energy range up to 600 keV. The novel dual-FOV aperture system allows selective imaging with two configurations: a 28-cm diameter wide FOV suitable for dual-leg or scout imaging and a 16-cm diameter high-resolution, and high-sensitivity (HR-HS) FOV designed for single-leg or focused imaging. Utilizing uniform phantoms, resolution phantoms, and multi-tracer phantoms, we experimentally assessed the system’s sensitivity, spatial resolution, and multi-tracer imaging capabilities. Spatial resolutions were approximately 6 mm in HR-HS-FOV mode and between 8 mm to 10 mm in wide-FOV mode. Peak-to-Valley ratios, indicative of image clarity, improved with enhanced DOI resolutions, rising from 1.03 to 1.22. The system’s ability to perform multi-tracer imaging, essential for deriving multifunctional molecular information, further highlights its potential to significantly enhance diagnostic accuracy for PVD.

This paper establishes a Freidlin-Wentzell large deviation principle for stochastic differential equations(SDEs) under locally weak monotonicity conditions and Lyapunov conditions. We illustrate the main result of the paper by showing that it can be applied to SDEs with non-Lipschitzian coefficients, which can not be covered in the existing literature. These include the interesting biological models like stochastic Duffing-van der Pol oscillator model, stochastic SIR model, etc.

The nominally trigonal, pseudo-Jahn-Teller (PJT)-active, S = 1/2 N2-bound complexes, P3EM(N2), M = Fe, Co, with three in-plane phosphine ligands and axial donors, E = Si, B, C, include functional nitrogenase models that catalyze the reduction of N2 to NH3. We applied EPR, 31P ENDOR spectroscopy, and DFT computations to characterize the PJT-induced distortions of four selected P3EM(N2), revealing how the metal ion and axial ligand E together tune both PJT dynamics, as revealed by 31P ENDOR and N2 activation, as indicated by a decrease in N≡N stretching frequency, ν(N≡N). P3SiFe(N2), P3BFe(N2)-, and P3BCo(N2) each exhibit a single 31P isotropic hyperfine coupling, revealing dynamic pseudorotation of the PJT distortion, producing averaged C3 symmetry with equivalent phosphine ligands. Conversely, P3CCo(N2)+ exhibits a static PJT distortion directed toward a phosphine bonded to a dx2-y2 SOMO lobe, leading to the exceptional isotropic 31P coupling aiso(31P) = +250 MHz, leaving two phosphines, aiso(31P) ≈ -20 MHz. Importantly, minimization of metal 'doming' out of the P3CCo(N2)+ phosphine plane toward N2 imposed by C-Co bond 'inelasticity' yields the longest M-N2 bond and the least-activated N2, as measured by ν(N≡N). Comparisons reveal an unrecognized correlation among PJT distortion, M-E bond elasticity, and N2 activation, providing guidelines for designing bioinspired N2-reduction catalysts.

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.

Mn2+ coordinated by orthophosphate (Pi), metabolites, or peptides acts as a superoxide dismutase (SOD), and these Mn antioxidant complexes are universally accumulated in extremely radiation- resistant cell types across the tree of life. This behavior prompted design of decapeptide DP1 (DEHGTAVMLK) as a Mn2+ ligand, and development of a highly potent Mn2+- antioxidant (MDP) containing [Pi] = 25 mM, and [DP1] = 3 mM, the ratio found in the radioresistant bacterium Deinococcus radiodurans, with [Mn2+] = 1 mM. MDP is an exceptional antioxidant, both in vitro and in vivo, and has reinvigorated the development of radiation- inactivated whole- cell vaccines. This study investigates the nature of the active Mn2+ complex in MDP. We measure the affinity of DP1 for the substitutionally labile Mn2+ ion using isothermal- titration calorimetry (ITC) and use changes in the Mn2+ solution EPR spectrum to determine affinities of Mn2+ for DP1 and for Pi, and to monitor Mn2+ ligation while titrated with the fixed Pi/DP1 ratio of MDP, 25/3, using ENDOR/ESEEM to characterize DP1 ligation to Mn2+. In parallel, 1 H NMR of DP1 was used to monitor binding interactions between Pi and DP1, and DP1 binding to the diamagnetic Ca2+. We report: i) DP1 forms an extremely weak, dynamic Mn2+ complex (Ka approximate to 40 M-1) ii) Mn2+ binds Pi much more strongly (Ka approximate to 390 M-1) as shown previously, but iii) DP1 and Pi jointly bind to Mn2+ in MDP to form a ternary Mn2+ (Pi) (DP1) complex with greater formation- constant than Pi alone (K a app approximate to 670 M-1). It is this ternary complex that is the superb antioxidant in MDP.

The generation of an active [FeFe]-hydrogenase requires the synthesis of a complex metal center, the H-cluster, by three dedicated maturases: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. A key step of [FeFe]-hydrogenase maturation is the synthesis of the dithiomethylamine (DTMA) bridging ligand, a process recently shown to involve the aminomethyl-lipoyl-H-protein from the glycine cleavage system, whose methylamine group originates from serine and ammonium. Here we use functional assays together with electron paramagnetic resonance and electron-nuclear double resonance spectroscopies to show that serine or aspartate together with their respective ammonia-lyase enzymes can provide the nitrogen for DTMA biosynthesis during in vitro [FeFe]-hydrogenase maturation. We also report bioinformatic analysis of the hyd operon, revealing a strong association with genes encoding ammonia-lyases, suggesting important biochemical and metabolic connections. Together, our results provide evidence that ammonia-lyases play an important role in [FeFe]-hydrogenase maturation by delivering the ammonium required for dithiomethylamine ligand synthesis.

Deep convolutional neural networks (CNNs) have achieved impressive success in enhancing the quality of compressed images/videos. These approaches mostly obtain the noise level in advance and train multiple architecture-identical models for enhancement on images/videos of known levels of noise. It largely hinders their practical applications where the noise level is unknown and resource is limited. To practically perform quality enhancement, we propose a novel blind quality enhancement framework for compressed video (BQEV), which utilizes a single network to conduct enhancement on videos compressed at various and unknown quality parameters (QPs). Since there exists feature similarity and difference among videos compressed at multiple QPs, BQEV utilizes this prior to efficiently handle enhancement on videos compressed at blind QPs, which consists of progressive feature extraction and QP-adaptive feature fusion subnets. They utilize temporal information and feature similarity to progressively extract valuable features and further employ the feature difference to conduct reasonable QP-adaptive feature fusion and quality enhancement, respectively. In the progressive feature extraction subnet, we first design a quality rank module to assign more attention to higher-quality frames for efficient utilization of temporal information, then propose a progressive extraction module to further extract features from different QPs. In the QPadaptive feature fusion subnet, we develop a quality estimation module to guide reasonable feature fusion of these extracted progressive features for stable and promising enhancement results on multiple QPs. Experimental results demonstrate that BQEV achieves 0.31 – 0.69 dB PSNR improvement compared with videos compressed at various QPs, outperforming state-ofthe-art approaches.

H-1/2 and C-13 hyperfine coupling constants to 5 ' -deoxyadenosyl (5 '-dAdo center dot) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2p pi orbital of the sp(2) C5 '-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5 '-dAdo center dot free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5 '-dAdo center dot is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant Fe-57, C-13, N-15, and H-2 hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S](2+ )cluster cogenerated with 5 '-dAdo center dot during homolytic cleavage of cluster-bound SAM. The origin of the Fe-57 couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5 '(dAdo center dot) to labeled-methionine as structural constraints yields a model for active-site positioning of 5 '-dAdo center dot with a short, nonbonded C5 '-Fe distance (similar to 3 angstrom). This distance involves substantial motion of 5 '-dAdo center dot toward the unique Fe of the [4Fe-4S](2+) cluster upon S-C(5 ') bond-cleavage, plausibly an initial step toward formation of the Fe-C5 ' bond of the organometallic complex, Omega, the central intermediate in catalysis by radical-SAM enzymes.

In this paper, we develop a new method to obtain the accessibility of stochastic partial differential equations driven by additive pure jump noise. An important novelty of this paper is to allow the driving noises to be degenerate. As an application, for the first time, we obtain the accessibility of a class of stochastic equations driven by pure jump (possibly degenerate) noise, including stochastic 2D Navier-Stokes equations, stochastic Burgers equations, stochastic singular p p -Laplace equations, and stochastic fast diffusion equations. As a further application, we establish the ergodicity of stochastic singular p p -Laplace equations and stochastic fast diffusion equations driven by additive pure jump noise, and we remark that the driving noises could be Compound Poisson processes or Lévy processes with heavy tails.

Radical enzymes, including the evolutionarily ancient glycyl radical enzyme (GRE) family, catalyze chemically challenging reactions that are involved in a myriad of important biological processes. All GREs possess an essential, conserved backbone glycine that forms a stable, catalytically essential α-carbon radical. Through close examination of the GRE family, we unexpectedly identified hundreds of noncanonical GRE homologs that encode either an alanine, serine, or threonine in place of the catalytic glycine residue. Contrary to a long-standing belief, we experimentally demonstrate that these aminoacyl radical enzymes (AAREs) form stable α-carbon radicals on the three cognate residues when activated by partner activating enzymes. The previously unrecognized AAREs are widespread in microbial genomes, highlighting their biological importance and potential for exhibiting new reactivity. Collectively, these studies expand the known radical chemistry of living systems while raising questions about the evolutionary emergence of the AAREs.

Considering irreducibility is fundamental for studying the ergodicity of stochastic dynamical systems. In this paper, we establish the irreducibility of stochastic complex Ginzburg-Laudau equations driven by pure jump noise. Our results are dimension free and the conditions placed on the driving noises are very mild. A crucial role is played by criteria developed by the authors of this paper and T. Zhang for the irreducibility of stochastic equations driven by pure jump noise. As an application, we obtain the ergodicity of stochastic complex Ginzburg-Laudau equations. We remark that our ergodicity result covers the weakly dissipative case with pure jump degenerate noise.

With the development of Industry 4.0, many manufacturers are moving in the direction of automation and intelligence. Drug package inspection quality testing methods have also changed from traditional manual inspection to machine vision inspection. However, due to the long exposure time or vibration of the camera, the images are blurred, which will seriously affect the accuracy of the defect detection. We propose a deblur model with higher level of image restoration and more lightweight. Using reserving and merging blocks to replace the original reserving and merging blocks, which can be used in the inference phase to turn ResBlocks into equivalent straight structures using reserving and merging operations, making the network lighter. The pyramid squeeze attention module is also incorporated to enable the model to efficiently extract finer-grained multi-specimen scale spatial information, thus making more efficient use of feature space information. Finally, a drug package dataset was produced and the model was trained to obtain good deblurring results. The comparative experiments confirmed that the model has better image reduction and excellent reduction speed, and can be further cited in production inspection pipelines to improve detection accuracy.

Named entity disambiguation (NED) is a fundamental task in NLP. Although numerous methods have been proposed for NED in recent years, they ignore the fact that a lot of real-world corpora are diachronic by nature, such as historical documents or news articles, which vary greatly in time. As a consequence, most current methods fail to fully exploit the temporal information inside the corpora and knowledge bases. To address the issue, we propose a novel model which integrates temporal feature into pretrained language model to make our model aware of time and a new sample re-weighting scheme for diachronic NED which penalizes highly-frequent mention-entity pairs to improve performance on rare and unseen entities. We present WikiCMAG and WikiSM, two new NED datasets annotated on ancient Chinese historical records. Experiments show that our model outperforms existing methods by large margins, proving the effectiveness of integrating diachronic information and our re-weighting schema. Our model also gains competitive performance on out-of-distribution (OOD) settings. WikiSM is publicly available at https://github.com/PKUDHC/WikiSM .

We constructed a prototype positron emission tomography (PET) system and experimentally evaluated large-volume 3-D cadmium zinc telluride (CZT) detectors for potential use in Compton-enhanced PET imaging. The CZT spectrometer offers sub-0.5-mm spatial resolution, an ultrahigh energy resolution (~1% @ 511 keV), and the capability of detecting multiple gamma-ray interactions that simultaneously occurred. The system consists of four CZT detector panels with a detection area of around 4.4 cm × 4.4 cm. The distance between the front surfaces of the two opposite CZT detector panels is ~80 mm. This system allows us to detect coincident annihilation photons and Compton interactions inside the detectors and then, exploit Compton kinematics to predict the first Compton interaction site and reject chance coincidences. We have developed a numerical integration technique to model the near-field Compton response that incorporates Doppler broadening, detector's finite resolutions, and the distance between the first and second interactions. This method was used to effectively reject random and scattered coincidence events. In the preliminary imaging studies, we have used point sources, line sources, a custom-designed resolution phantom, and a commercial image quality (IQ) phantom to demonstrate an imaging resolution of approximately 0.75 mm in PET images, and Compton-based enhancement.