A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA.

Nucleotide excision repair functions to protect genome integrity, and ongoing studies using excision repair sequencing (XR-seq) have contributed to our understanding of how cells prioritize repair across the genome. In this method, the products of excision repair bearing damaged DNA are captured, sequenced, and then mapped genome-wide at single-nucleotide resolution. However, reagent requirements and complex procedures have limited widespread usage of this technique. In addition to the expense of these reagents, it has been hypothesized that the immunoprecipitation step using antibodies directed against damaged DNA may introduce bias in different sequence contexts. Here, we describe a newly developed adaptation called dA-tailing and adaptor ligation (ATL)-XR-seq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 3'-dA-tailing and 5'-adapter ligation instead of the original 5'- and 3'-dual adapter ligation. This new approach avoids adapter dimer formation during subsequent PCR, omits inefficient and time-consuming purification steps, and is very sensitive. In addition, poly(dA) tail length heterogeneity can serve as a molecular identifier, allowing more repair hotspots to be mapped. Importantly, a comparison of both repair mapping methods showed that no major bias is introduced by the anti-UV damage antibodies used in the original XR-seq procedure. Finally, we also coupled the described dA-tailing approach with quantitative PCR in a new method to quantify repair products. These new methods provide powerful and user-friendly tools to qualitatively and quantitatively measure excision repair.

Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

Proposal for complete characterization of attosecond pulses from relativistic plasmas.

In this study, we propose two full-optical-setup and single-shot measurable approaches for complete characterization of attosecond pulses from surface high harmonic generation (SHHG): SHHG-SPIDER (spectral phase interferometry for direct electric field reconstruction) and SHHG-SEA-SPIDER (spatially encoded arrangement for SPIDER). 1D- and 2D-EPOCH PIC (particle-in-cell) simulations were performed to generate the attosecond pulses from relativistic plasmas under different conditions. Pulse trains dominated by single isolated peak as well as complex pulse train structures are extensively discussed for both methods, which showed excellent accuracy in the complete reconstruction of the attosecond field with respect to the direct Fourier transformed result. Kirchhoff integral theorem has been used for the near-to-far-field transformation. This far-field propagation method allows us to relate these results to potential experimental implementations of the scheme. The impact of comprehensive experimental parameters for both apparatus, such as spectral shear, spatial shear, cross-angle, time delay, and intensity ratio between the two replicas has been investigated thoroughly. These methods are applicable to complete characterization for SHHG attosecond pulses driven by a few to hundreds of terawatts femtosecond laser systems.

IFNb of black carp functions importantly in host innate immune response as an antiviral cytokine.

Type I interferons (IFN-Is) play an important role in the antiviral immune response in teleost fishes. In this study, one type I interferon (bcIFNb) from black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of bcIFNb gene consists of 806 nucleotides and the predicted bcIFNb protein contains 188 amino acids. Basing on the cysteine number and evolutionary position, bcIFNb was classified into group II type I IFN. q-PCR analysis demonstrated that bcIFNb mRNA level varied in vivo and ex vivo in response to different stimuli. bcIFNb was detected in both the whole cell lysate and the supernatant media of HEK293T cells or EPC cells transfected with bcIFNb through immunoblot assay. IFN stimulated genes (ISGs) were greatly upregulated when the host cells were treated with the bcIFNb-containing conditioned media. EPC cells showed greatly enhanced antiviral ability when the cells were transfected with bcIFNb or treated with the bcIFNb-containing conditioned media before GCRV or SVCV infection. Glycosidase digestion analysis determined that bcIFNb was modified with N-linked glycosylation, which occurred on the Asn (N) of 92 site of this cytokine. The un-glycosylated mutant bcIFNb-N92Q presented the similar antiviral ability as that of wild type bcIFNb, which demonstrated that N-linked glycosylation did not contribute directly to the antiviral property of this fish cytokine.

Black carp STING functions importantly in innate immune defense against RNA virus.

Stimulator of interferon genes (STING) is a central and multifaceted mediator in the innate immune response of higher vertebrates. To explore its role in teleost fish, the STING homolog of black carp (Mylopharyngodon piceus) (bcSTING) has been cloned and characterized in this paper. bcSTING transcription in Mylopharyngodon piceus fin (MPF) cells increased remarkably in response to GCRV and SVCV infection, or poly (I:C) stimulation. bcSTING migrated around 42 KDa in immunoblot assay and was identified as a cytosolic protein locating on ER majorly through immunofluorescence staining. Under condition of SVCV/GCRV infection or poly (I:C) stimulation, the subcellular distribution of bcSTING majorly displayed on mitochondria, which overlapped with that of bcMAVS. HA-bcSTING instead of bcSTING-HA presented strong IFN-inducing activity in reporter assay and antiviral ability against both SVCV and GCRV in plaque assay. Site mutation of serine (S) on C-terminus of bcSTING demonstrated that both S371 and S379 were crucial for its mediated signaling. Taken together, our study support the conclusion that bcSTING plays an important role in host innate immune defense against RNA virus such as SVCV and GCRV, in which its C-terminus functions crucially.

Vortex laser beam generation from laser interaction with azimuthal plasma phase slab at relativistic intensities.

It is shown theoretically and by simulation that a Gaussian laser beam of relativistic intensity interacting with a uniform-thickness plasma slab of azimuthally varying density can acquire orbital angular momentum (OAM). During the interaction, the laser ponderomotive force and the charge-separation force impose a torque on the plasma particles. The affected laser light and plasma ions gain oppositely directed axial OAM, but the plasma electrons remain almost OAM free. High OAM conversion efficiency is achieved due to the strong azimuthal electromagnetic energy flow during the laser phase modulation. The present scheme should provide useful reference for applications requiring relativistic-intense vortex light.

Efficient copper(II)-catalyzed addition of activated methylene compounds to alkenes.

Efficient regioselective addition of beta-diketones to styrenes, norbornene, cyclic enol ether, and diene has been realized by means of copper(II) triflate as the catalyst. The solvent effect is prominent on the reactions, and the desired addition products were obtained in good to excellent yields only in dioxane or ionic liquid [bmim]PF6. The mechanism suggests that copper(II) triflate activates the enolic O-H bond of a beta-diketone substrate to initiate the addition reaction.

Magnetic-field generation and electron-collimation analysis for propagating fast electron beams in overdense plasmas.

An analytical fluid model is proposed for artificially collimating fast electron beams produced in the interaction of ultraintense laser pulses with specially engineered low-density-core-high-density-cladding structure targets. Since this theory clearly predicts the characteristics of the spontaneously generated magnetic field and its dependence on the plasma parameters of the targets transporting fast electrons, it is of substantial relevance to the target design for fast ignition. The theory also reveals that the rapid changing of the flow velocity of the background electrons in a transverse direction (perpendicular to the flow velocity) caused by the density jump dominates the generation of a spontaneous interface magnetic field for these kinds of targets. It is found that the spontaneously generated magnetic field reaches as high as 100 MG, which is large enough to collimate fast electron transport in overdense plasmas. This theory is also supported by numerical simulations performed using a two-dimensional particle-in-cell code. It is found that the simulation results agree well with the theoretical analysis.

Ruthenium(III) chloride catalyzed efficient synthesis of unsymmetrical diorganyl selenides via cleavage of dibenzyl and diphenyl diselenides in the presence of zinc.

[equation: see text] An efficient one-pot route to unsymmetrical diorganyl selenides has been developed by ruthenium(III) chloride catalyzed reactions of dibenzyl or diphenyl diselenides with alkyl halides in the presence of zinc. Organic iodides, bromides, and activated chlorides underwent the reactions efficiently. Unreactive organic chlorides also underwent the same type of selenation with sodium bromide as the additive.