Shisheng Li

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Vertically aligned carbon nanotubes (VACNTs) are grown directly on a free-standing graphene paper (GP). The desirable carrier transport ability of the VACNTs, good conductivity and mechanical properties of the GP, and strong bonding between the VACNTs and the GP endow the hybrid structure with superior performance when utilized as the electrodes of lithium-ion batteries and dye-sensitized solar cells. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Chemical vapour deposition of two-dimensional materials typically involves the conversion of vapour precursors to solid products in a vapour-solid-solid mode. Here, we report the vapour-liquid-solid growth of monolayer MoS2, yielding highly crystalline ribbons with a width of few tens to thousands of nanometres. This vapour-liquid-solid growth is triggered by the reaction between MoO3 and NaCl, which results in the formation of molten Na-Mo-O droplets. These droplets mediate the growth of MoS2 ribbons in the 'crawling mode' when saturated with sulfur. The locally well-defined orientations of the ribbons reveal the regular horizontal motion of the droplets during growth. Using atomic-resolution scanning transmission electron microscopy and second harmonic generation microscopy, we show that the ribbons are grown homoepitaxially on monolayer MoS2 with predominantly 2H- or 3R-type stacking. Our findings highlight the prospects for the controlled growth of atomically thin nanostructure arrays for nanoelectronic devices and the development of unique mixed-dimensional structures.

We present a metal-catalyst-free CVD process for the high-efficiency growth of single-walled carbon nanotubes (SWNTs) on surface. By applying a 30-nm-thick SiO2 sputtering deposited Si or Si/SiO2 wafer as substrate and CH4 as a carbon source, dense and uniform SWNT networks with high quality can be obtained without the presence of any metal species. Moreover, a simple patterned growth approach, using a scratched Si/SiO2 wafer as substrate, is also presented for the growth of SWNTs with good position controllability. Our finding of the growth of SWNTs via a metal-catalyst-free process will provide valuable information for understanding the growth mechanism of SWNTs in-depth, which accordingly will facilitate the controllable synthesis and applications of carbon nanotubes.

Fruits are the defining feature of angiosperms, likely have contributed to angiosperm successes by protecting and dispersing seeds, and provide foods to humans and other animals, with many morphological types and important ecological and agricultural implications. Rosaceae is a family with ∼3000 species and an extraordinary spectrum of distinct fruits, including fleshy peach, apple, and strawberry prized by their consumers, as well as dry achenetum and follicetum with features facilitating seed dispersal, excellent for studying fruit evolution. To address Rosaceae fruit evolution and other questions, we generated 125 new transcriptomic and genomic datasets and identified hundreds of nuclear genes to reconstruct a well-resolved Rosaceae phylogeny with highly supported monophyly of all subfamilies and tribes. Molecular clock analysis revealed an estimated age of ∼101.6 Ma for crown Rosaceae and divergence times of tribes and genera, providing a geological and climate context for fruit evolution. Phylogenomic analysis yielded strong evidence for numerous whole genome duplications (WGDs), supporting the hypothesis that the apple tribe had a WGD and revealing another one shared by fleshy fruit-bearing members of this tribe, with moderate support for WGDs in the peach tribe and other groups. Ancestral character reconstruction for fruit types supports independent origins of fleshy fruits from dry-fruit ancestors, including the evolution of drupes (e.g., peach) and pomes (e.g., apple) from follicetum, and drupetum (raspberry and blackberry) from achenetum. We propose that WGDs and environmental factors, including animals, contributed to the evolution of the many fruits in Rosaceae, which provide a foundation for understanding fruit evolution.

The recent discovery of a Weyl semimetal in TaAs offers the first Weyl fermion observed in nature and dramatically broadens the classification of topological phases. However, in TaAs it has proven challenging to study the rich transport phenomena arising from emergent Weyl fermions. The series MoxW1-xTe2 are inversion-breaking, layered, tunable semimetals already under study as a promising platform for new electronics and recently proposed to host Type II, or strongly Lorentz-violating, Weyl fermions. Here we report the discovery of a Weyl semimetal in MoxW1-xTe2 at x=25%. We use pump-probe angle-resolved photoemission spectroscopy (pump-probe ARPES) to directly observe a topological Fermi arc above the Fermi level, demonstrating a Weyl semimetal. The excellent agreement with calculation suggests that MoxW1-xTe2 is a Type II Weyl semimetal. We also find that certain Weyl points are at the Fermi level, making MoxW1-xTe2 a promising platform for transport and optics experiments on Weyl semimetals.

Two-dimensional (2D) transition metal dichalcogenide (TMD) nanosheets have attracted considerable attention owing to their diverse properties and great potential in a wide range of applications. In order to further tune their properties and then broaden their application domain, large efforts have been devoted into engineering the structures of 2D TMD nanosheets at atomic scale, especially the alloying technology. Alloying different 2D TMD nanosheets into 2D alloys not only offers the opportunities to fine-tune their physical/chemical properties, but also opens up some unique properties, which are highly desirable for wide applications including electronics, optoelectronics and catalysis. This review summarizes the recent progress in the preparation, characterization and applications of 2D alloyed TMD nanosheets.

In view of their low immunogenicity, biomimetic internal environment, tissue- and organ-like physicochemical properties, and functionalization potential, decellularized extracellular matrix (dECM) materials attract considerable attention and are widely used in tissue engineering. This review describes the composition of extracellular matrices and their role in stem-cell differentiation, discusses the advantages and disadvantages of existing decellularization techniques, and presents methods for the functionalization and characterization of decellularized scaffolds. In addition, we discuss progress in the use of dECMs for cartilage, skin, nerve, and muscle repair and the transplantation or regeneration of different whole organs (e.g., kidneys, liver, uterus, lungs, and heart), summarize the shortcomings of using dECMs for tissue and organ repair after refunctionalization, and examine the corresponding future prospects. Thus, the present review helps to further systematize the application of functionalized dECMs in tissue/organ transplantation and keep researchers up to date on recent progress in dECM usage.

Abstract Malignancy is a major public health problem and among the leading lethal diseases worldwide. Although the current tumor treatment methods have therapeutic effect to a certain extent, they still have some shortcomings such as poor water solubility, short half-life, local and systemic toxicity. Therefore, how to deliver therapeutic agent so as to realize safe and effective anti-tumor therapy become a problem urgently to be solved in this field. As a medium of information exchange and material transport between cells, exosomes are considered to be a promising drug delivery carrier due to their nano-size, good biocompatibility, natural targeting, and easy modification. In this review, we summarize recent advances in the isolation, identification, drug loading, and modification of exosomes as drug carriers for tumor therapy alongside their application in tumor therapy. Basic knowledge of exosomes, such as their biogenesis, sources, and characterization methods, is also introduced herein. In addition, challenges related to the use of exosomes as drug delivery vehicles are discussed, along with future trends. This review provides a scientific basis for the application of exosome delivery systems in oncological therapy. Graphical Abstract

Semiconducting single-walled carbon nanotubes (s-SWCNTs) with a mean diameter of 1.6 nm were synthesized on a large scale by using oxygen-assisted floating catalyst chemical vapor deposition. The oxygen introduced can selectively etch metallic SWCNTs in situ, while the sulfur growth promoter functions in promoting the growth of SWCNTs with a large diameter. The electronic properties of the SWCNTs were characterized by laser Raman spectroscopy, absorption spectroscopy, and field effect transistor measurements. It was found that the content of s-SWCNTs in the samples was highly sensitive to the amount of oxygen introduced. Under optimum synthesis conditions, enriched s-SWCNTs can be obtained in milligram quantities per batch.

Grain boundaries have a major effect on the physical properties of two-dimensional layered materials. Therefore, it is important to develop simple, fast and sensitive characterization methods to visualize grain boundaries. Conventional Raman and photoluminescence methods have been used for detecting grain boundaries; however, these techniques are better suited for detection of grain boundaries with a large crystal axis rotation between neighbouring grains. Here we show rapid visualization of grain boundaries in chemical vapour deposited monolayer MoS2 samples with multiphoton microscopy. In contrast to Raman and photoluminescence imaging, third-harmonic generation microscopy provides excellent sensitivity and high speed for grain boundary visualization regardless of the degree of crystal axis rotation. We find that the contrast associated with grain boundaries in the third-harmonic imaging is considerably enhanced by the solvents commonly used in the transfer process of two-dimensional materials. Our results demonstrate that multiphoton imaging can be used for fast and sensitive characterization of two-dimensional materials.

Exciton-plasmon coupling in hybrids of a monolayer transition metal dichalcogenide and Ag nanoparticles is investigated in the weak and strong coupling regimes. In the weak coupling regime, both absorption enhancement and the Purcell effect collectively modify the photoluminescence properties of the semiconductor. In the strong coupling regime, electromagnetically induced transparency dips are displayed, evidencing coherent energy exchange between excitons and plasmons.

Vapor transportation is the core process in growing transition-metal dichalcogenides (TMDCs) by chemical vapor deposition (CVD). One inevitable problem is the spatial inhomogeneity of vapors. The non-stoichiometric supply of transition-metal precursors and chalcogens leads to poor control in the products' location, morphology, crystallinity, uniformity and batch to batch reproducibility. The vapor-liquid-solid (VLS) growth method often involves molten precursors (e.g., non-volatile Na2MoO4) at growth temperatures higher than their melting points. The liquid Na2MoO4 can precipitate out solid MoS2 monolayers when saturated with sulfur vapor. Taking advantage of the VLS growth, we attained three kinds of important achievements: (i) a 4-inch-wafer-scale uniform growth of MoS2 flakes on SiO2/Si substrates, (ii) a 2-inch-wafer-scale growth of continuous MoS2 film with the grain size exceeding 100 μm on sapphire substrates, and (iii) a patterned (site-controlled) growth of MoS2 flakes and films. We clarified that the VLS growth thus paves a new way for the high-efficient and scalable synthesis of two-dimensional TMDC monolayers.

Substitutional doping has been proven to be an effective route to engineer band gap, transport characteristics, and magnetism in transition metal dichalcogenides. Herein, we demonstrate substitutional doping of monolayer tungsten disulfide (WS2) with Nb via the chemical vapor deposition technique. Scanning transmission electron microscopy confirms that Nb successfully substituted the W atom in the WS2 lattice. Moreover, photoluminescence indicates a significant red shift when different concentrations of Nb are introduced, in agreement with density functional theory calculations. Electrical measurements reveal the degenerate p-type semiconductor behavior of Nb-doped WS2 field-effect transistors. The successful synthesis of p-type WS2 in this study provides a promising method to expand the electronic and photonic engineering of two-dimensional materials.

Abstract Defects and their spatial distribution are crucial factors in controlling the electronic and optical properties of semiconductors. By using scanning transmission electron microscopy and electron energy loss spectroscopy, the type of impurities/defects in WS 2 subdomains with different optical properties is successfully assigned. A higher population of Cr impurities is found in the W‐terminated edge domain, while the S‐terminated domain contains more Fe impurities, in accordance with the luminescence characteristics of chemical‐vapor‐grown WS 2 of a hexagonal shape. In agreement with the first‐principles calculations, the domains with Cr substitutional dopants exhibit strong trion emission. Fe atoms tend to gather into trimer configuration and introduce deep acceptor levels which compensate the n‐type doping and suppress trion emission. It is also discovered that the domain with higher luminescence but smaller defect concentration tends to get oxidized more rapidly and degrade the 2D structure with many triangular holes. Excitons tend to accumulate at the edges of the oxidized triangular holes and results in enhanced PL emission. The findings indicate that choosing stable elements as dopant and controlling the number of specific edge structures within a crystal domain of 2D transitional metal dichalcogenides can be a new route to improve the optical properties of these materials.

Abstract Two‐dimensional (2D) transition metal dichalcogenides (TMDCs) with unique electrical properties are fascinating materials used for future electronics. However, the strong Fermi level pinning effect at the interface of TMDCs and metal electrodes always leads to high contact resistance, which seriously hinders their application in 2D electronics. One effective way to overcome this is to use metallic TMDCs or transferred metal electrodes as van der Waals (vdW) contacts. Alternatively, using highly conductive doped TMDCs will have a profound impact on the contact engineering of 2D electronics. Here, a novel chemical vapor deposition (CVD) using mixed molten salts is established for vapor–liquid–solid growth of high‐quality rhenium (Re) and vanadium (V) doped TMDC monolayers with high controllability and reproducibility. A tunable semiconductor to metal transition is observed in the Re‐ and V‐doped TMDCs. Electrical conductivity increases up to a factor of 10 8 in the degenerate V‐doped WS 2 and WSe 2 . Using V‐doped WSe 2 as vdW contact, the on‐state current and on/off ratio of WSe 2 ‐based field‐effect transistors have been substantially improved (from ≈10 –8 to 10 –5 A; ≈10 4 to 10 8 ), compared to metal contacts. Future studies on lateral contacts and interconnects using doped TMDCs will pave the way for 2D integrated circuits and flexible electronics.

Drought is an abiotic stress that affects plant growth, and lipids are the main economic factor in the agricultural production of oil crops. However, the molecular mechanisms of drought response function in lipid metabolism remain little known. In this study, overexpression (OE) of different copies of the drought response genes LEA3 and VOC enhanced both drought tolerance and oil content in Brassica napus and Arabidopsis. Meanwhile, seed size, membrane stability and seed weight were also improved in OE lines. In contrast, oil content and drought tolerance were decreased in the AtLEA3 mutant (atlea3) and AtVOC-RNAi of Arabidopsis and in both BnLEA-RNAi and BnVOC-RNAi B. napus RNAi lines. Hybrids between two lines with increased or reduced expression (LEA3-OE with VOC-OE, atlea3 with AtVOC-RNAi) showed corresponding stronger trends in drought tolerance and lipid metabolism. Comparative transcriptomic analysis revealed the mechanisms of drought response gene function in lipid accumulation and drought tolerance. Gene networks involved in fatty acid (FA) synthesis and FA degradation were up- and down-regulated in OE lines, respectively. Key genes in the photosynthetic system and reactive oxygen species (ROS) metabolism were up-regulated in OE lines and down-regulated in atlea3 and AtVOC-RNAi lines, including LACS9, LIPASE1, PSAN, LOX2 and SOD1. Further analysis of photosynthetic and ROS enzymatic activities confirmed that the drought response genes LEA3 and VOC altered lipid accumulation mainly via enhancing photosynthetic efficiency and reducing ROS. The present study provides a novel way to improve lipid accumulation in plants, especially in oil production crops.

Two-dimensional (2D) materials family with its many members and different properties has recently drawn great attention. Thanks to their atomic thickness and smooth surface, 2D materials can be constructed into heterostructures or homostructures in the fashion of out-of-plane perpendicular stacking or in-plane lateral stitching, resulting in unexpected physical and chemical properties and applications in many areas. In particular, 2D metal-semiconductor heterostructures or homostructures (MSHSs) which integrate 2D metals and 2D semiconductors, have shown great promise in future integrated electronics and energy-related applications. In this review, MSHSs with different structures and dimensionalities are first introduced, followed by several ways to prepare them. Their applications in electronics and optoelectronics, energy storage and conversion, and their use as platforms to exploit new physics are then discussed. Finally, we give our perspectives about the challenges and future research directions in this emerging field.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR–Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms. Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.

Pulmonary drug delivery has the advantages of being rapid, efficient, and well-targeted, with few systemic side effects. In addition, it is non-invasive and has good patient compliance, making it a highly promising drug delivery mode. However, there have been limited studies on drug delivery via pulmonary inhalation compared with oral and intravenous modes. This paper summarizes the basic research and clinical translation of pulmonary inhalation drug delivery for the treatment of diseases and provides insights into the latest advances in pulmonary drug delivery. The paper discusses the processing methods for pulmonary drug delivery, drug carriers (with a focus on various types of nanoparticles), delivery devices, and applications in pulmonary diseases and treatment of systemic diseases (e.g., COVID-19, inhaled vaccines, diagnosis of the diseases, and diabetes mellitus) with an updated summary of recent research advances. Furthermore, this paper describes the applications and recent progress in pulmonary drug delivery for lung diseases and expands the use of pulmonary drugs for other systemic diseases.

We propose a novel surface and interference coenhanced Raman scattering technique to dramatically enhance the Raman signal intensity of graphene by using a specifically designed substrate of Si capped with surface-active metal and oxide double layers (SMO). The total enhancement ratio can reach the order of 103 compared with the original Si substrate. Combining the visibility of graphene on the SMO substrate, we demonstrate that the tiny structure change and surface structure of graphene can be easily detected. This technique makes Raman spectroscopy a more powerful tool in the field of ultrasensitive characterization of graphene, isolated carbon nanotubes, and other film-like materials.

Chirality-controlled synthesis of single-walled carbon nanotubes (SWCNTs) is a prerequisite for their practical applications in electronic and optoelectronic devices. We report here a novel bimetallic CoPt catalyst for the selective growth of high quality SWCNTs with a narrow chirality distribution at relatively high temperatures of 800 °C and 850 °C using atmospheric pressure alcohol chemical vapor deposition. The addition of Pt into a Co catalyst forms a CoPt alloy and significantly reduces the diameters of the as-grown SWCNTs and narrows their chirality distributions.

Due to its outstanding performance, carbon nanotube (CNT) has shown great potentials as an enhancement material for composites. However, CNT tends to cluster together or form aggregates during fabrication process, it is a challenging task to effectively mix CNT into a metal matrix. In this study, a novel approach is proposed to disperse CNT into aluminum matrix by using silicon carbide particles (SiCp) as a “carrier”. Through chemical vapor deposition, CNT is grown on the surface of micro-sized SiCp forming a nano/micro-sized hybrid reinforcement, which is named as SiCp(CNT), and then they will be homogeneously distributed in the 6061Al matrix by conventional powder metallurgy process. Three types of SiCp(CNT), with SiCp sizes being 13 μm, 7 μm, 2.5 μm, were used for finding out the effect of SiCp size on the morphology of SiCp(CNT), the microstructure and mechanical properties of the obtained composite. The most significant improvement in tensile properties can be found for the case with the SiCp size to be 7 μm, which has ductility of 8.5%, Young's modulus of 98 GPa, and tensile strength of 428 MPa. This remarkable strengthening effect is attributed to the increase of the “punched zone” size with the presence of CNT on the SiCp surface.

Abstract Laryngeal cancer is one of the most common head and neck malignant tumors and is commonly resistant to X‐ray‐based radiotherapy. NF ‐ κ B interacting lncRNA ( NKILA ) has been reported to serve as a tumor suppressor in several cancers through combining with NF ‐ κ B: I κ B complex thereby inhibiting NF ‐ κ B activation. Herein, we demonstrated a low NKILA expression in laryngeal cancer and its correlation with shorter overall survival in patients with laryngeal cancer. NKILA serves as a tumor suppressor in laryngeal cancer by suppressing laryngeal cancer cell viability and migration, whereas promoting cell apoptosis; NKILA knockdown reverses the cytotoxicity of X‐ray radiation on laryngeal cancer cells through combining with NF ‐ κ B: I κ B complex to inhibit I κ B phosphorylation, inhibit p65 nuclear translocation, and finally inhibit NF ‐ κ B activation. NF ‐ κ B binds to the promoter region of NKILA to activate its transcriptional activity, upregulated NKILA then inhibits I κ B phosphorylation and NF ‐ κ B activation, thus forming a negative feedback loop to sensitize laryngeal cancer cell to X‐ray radiation. In conclusion, NKILA can serve as a promising agent of enhancing the cytotoxicity of X‐ray radiation on laryngeal cancer and addressing the radioresistance of laryngeal cancer.

Effective thermal management is becoming increasingly important for high power density electronic devices, which requires materials with high thermal conductivity. A novel graphite film/aluminum composite material with ultrahigh thermal conductivity was fabricated by vacuum hot pressing process to meet this requirement. The effects of fabrication parameters on microstructures and thermal conductivities of these composites were investigated. As a result, the composites with graphite volume fraction of 17.4–69.4% sintered at 655 °C/45 MPa for 100 min exhibit in-plane thermal conductivities of 380–940 W/mK, over 90% of the predictions by rule of mixture. These composites possess higher in-plane thermal conductivities than graphite flake/aluminum composites due to well-controlled graphite orientation and fabrication parameters, which indicates that these composites are promising materials for effective thermal management.

Spt5, a transcription elongation factor, and Rpb4, a subunit of RNA polymerase II (RNAP II) that forms a subcomplex with Rpb7, play important roles in transcription elongation and repression of transcription coupled DNA repair (TCR) in eukaryotic cells. How Spt5 physically interacts with RNAP II, and if and/or how Spt5 and Rpb4/7 coordinate to achieve the distinctive functions have been enigmatic. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, a photoreactive cross-linker, we mapped interactions between Spt5 and RNAP II in Saccharomyces cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.

Recent findings about ultrahigh thermoelectric performances in SnSe single crystals have stimulated research on this binary semiconductor material. Furthermore, single-layer SnSe is an interesting analogue of phosphorene, with potential applications in two-dimensional (2D) nanoelectronics. Although significant advances in the synthesis of SnSe nanocrystals have been made, fabrication of well-defined large-sized single-layer SnSe flakes in a facile way still remains a challenge. The growth of single-layer rectangular SnSe flakes with a thickness of ~6.8 Å and lateral dimensions of about 30 µm × 50 µm is demonstrated by a two-step synthesis method, where bulk rectangular SnSe flakes were synthesized first by a vapor transport deposition method followed by a nitrogen etching technique to fabricate single-layer rectangular SnSe flakes in an atmospheric pressure system. The as-obtained rectangular SnSe flakes exhibited a pure crystalline phase oriented along the a-axis direction. Field-effect transistor devices fabricated on individual single-layer rectangular SnSe flakes using gold electrodes exhibited p-doped ambipolar behavior and a hole mobility of about 0.16 cm2 V−1 s−1. This two-step fabrication method can be helpful for growing other similar 2D large-sized single-layer materials.

Shape engineering plays a crucial role in the application of two-dimensional (2D) layered metal dichalcogenide (LMD) crystalline materials in terms of physical and chemical property modulation. However, controllable growth of 1T phase tin disulfide (SnS2) with multifarious morphologies has rarely been reported and remains challenging. Herein, we report a direct synthesis of large-size, uniform, and atomically thin 1T-SnS2 with multiple morphologies by adding potassium halides via a facile chemical vapor deposition process. A variety of morphologies, i.e., from hexagon, triangle, windmill, and dendritic to coralloid, corresponding to fractal dimensions from 1.01 to 1.81 are accurately controlled by growth conditions. Moreover, the Sn concentration controls the morphology change of SnS2. The edge length of the SnS2 dendritic flake can grow larger than 500 μm in 5 min. Potassium halides can significantly reduce the surface migration barrier of the SnS2 cluster and enhance the SnS2 adhesion force with substrate to facilitate efficient high in-plane growth of monolayer SnS2 compared to sodium halides by density functional theory calculations. More branched SnS2 with higher fractal dimension provides more active sites for enhancing hydrogen evolution reactions. Importantly, we prove that potassium halides are preferable for 1T-phase LMDs structures, while sodium halides are more suitable for 2H-phase materials. The growth mechanism proposed here provides a general approach for controllable-phase synthesis of 2D LMD crystals and related heterostructures. Shape engineering of 2D materials also provides a strategy to tune LMD properties for demanding applications.

Abstract Black phosphorus (BP), as a fast emerging 2D material, shows promising potential in near‐infrared (NIR) photodetection owing to its relatively small direct thickness‐dependent bandgaps. However, the poor NIR absorption due to the atomically thin nature strongly hinders the practical application. In this study, it is demonstrated that surface functionalization of Ag nanoclusters on 2D BP can induce an abnormal NIR absorption at ≈746 nm, leading to ≈35 (138) times enhancement in 808 (730) nm NIR photoresponse for BP‐based field‐effect transistors. First‐principles calculations reveal that localized bands are introduced into the bandgap of BP, serving as the midgap states, which create new transitions to the conduction band of BP and eventually lead to the abnormal absorption. This work provides a simple yet effective method to dramatically increase the NIR absorption of BP, which is crucial for developing high‐performance NIR optoelectronic devices.

Abstract Crystal phase control still remains a challenge for the precise synthesis of 2D layered metal dichalcogenide (LMD) materials. The T′ phase structure has profound influences on enhancing electrical conductivity, increasing active sites, and improving intrinsic catalytic activity, which are urgently needed for enhancing hydrogen evolution reaction (HER) activity. Theoretical calculations suggest that metastable T′ phase 2D Sn 1− x W x S 2 alloys can be formed by combining W with 1T tin disulfide (SnS 2 ) as a template to achieve a semiconductor‐to‐metallic transition. Herein, 2D Sn 1− x W x S 2 alloys with varying x are prepared by adjusting the molar ratio of reactants via hydrothermal synthesis, among which Sn 0.3 W 0.7 S 2 displays a maximum of concentration of 81% in the metallic phase and features a distorted octahedral‐coordinated metastable 1T′ phase structure. The obtained 1T′‐Sn 0.3 W 0.7 S 2 has high intrinsic electrical conductivity, lattice distortion, and defects, showing a prominently improved HER catalytic performance. Metallic Sn 0.3 W 0.7 S 2 coupled with carbon black exhibits at least a 215‐fold improvement compared to pristine SnS 2 . It has excellent long‐term durability and HER activity. This work reveals a general phase transition strategy by using T phase materials as templates and merging heteroatoms to achieve synthetic metastable phase 2D LMDs that have a significantly improved HER catalytic performance.

Growth of large-area, uniform, and high-quality monolayer transition-metal dichalcogenides (TMDs) for practical and industrial applications remains a long-standing challenge. The present study demonstrates a modified predeposited chemical vapor deposition (CVD) process by employing an annealing procedure before sulfurization, which helps in achieving large-area, highly uniform, and high-quality TMDs on various substrates. The annealing procedure resulted in a molten liquid state of the precursors in the CVD process, which not only facilitated a uniform redistribution of the precursor on the substrate (avoid the aggregation) because of the uniform redistribution of the liquid precursor on the substrate but more importantly avoided the undesired multilayer growth via the self-limited lateral supply precursors mechanism. A 2 in. uniform and continuous monolayer WS2 film has been synthesized on the SiO2/Si substrate. Moreover, uniform monolayer WS2 single crystals can be prepared on more general and various substrates including sapphire, mica, quartz, and Si3N4 using the same growth procedure. Besides, this growth mechanism can be generalized to synthesize other monolayer TMDs such as MoS2 and MoS2/WS2 heterostructures. Hence, the present method provides a generalized attractive strategy to grow large-area, uniform, single-layer two-dimensional (2D) materials. This study has significant implications in the advancement of batch production of various 2D-material-based devices for industrial and commercial applications.

Abstract The light–matter interaction in materials is of remarkable interest for various photonic and optoelectronic applications, which is intrinsically determined by the bandgap of the materials involved. To extend the applications beyond the bandgap limit, it is of great significance to study the light–matter interaction below the material bandgap. Here, we report the ultrafast transient absorption of monolayer molybdenum disulfide in its sub-bandgap region from ~0.86 µm to 1.4 µm. Even though this spectral range is below the bandgap, we observe a significant absorbance enhancement up to ~4.2% in the monolayer molybdenum disulfide (comparable to its absorption within the bandgap region) due to pump-induced absorption by the excited carrier states. The different rise times of the transient absorption at different wavelengths indicate the various contributions of the different carrier states (i.e., real carrier states in the short-wavelength region of ~<1 µm, and exciton states in the long wavelength region of ~>1 µm). Our results elucidate the fundamental understanding regarding the optical properties, excited carrier states, and carrier dynamics in the technologically important near-infrared region, which potentially leads to various photonic and optoelectronic applications (e.g., excited-state-based photodetectors and modulators) of two-dimensional materials and their heterostructures beyond their intrinsic bandgap limitations.

Surface water environment is an important repository of antibiotics and antibiotic resistance genes (ARGs). It is of great significance to study the occurrence and distribution of antibiotics and ARGs in surface water environment. In this study, the Liaohe River Basin, China was taken as the study area, and the concentrations of antibiotics and ARGs in water were investigated by HPLC-MS/MS and HT-qPCR. The results showed that a total of 53 antibiotics of 6 types were detected in water, and the pollution level was at ND~331.64 ng/L, where PCG was the highest. Totally 164 ARGs and 10 mobile genetic elements (MGEs) were detected in the water, and the absolute abundances were at 2.18 × 104~3.95 × 107 copies/L and 1.82 × 105–3.78 × 107 copies/L, respectively. The multidrug and aminoglycoside were the dominant ARG types. Amoxicillin, erythromycin, anhydroerythromycin and ofloxacin posed certain ecological risks for sensitive aquatic organisms. In spatial distribution, the pollution of antibiotics and ARGs in the Daliao River system was higher than that in the Liaohe River system. There was a significant positive correlation between total concentrations of antibiotics and total relative abundance of ARGs (r = 0.66, p < 0.05). The co-occurrence of multiple antibiotics promoted the pollution and spread of ARGs. In addition, the total relative abundance of MGEs and ARGs showed a significant positive correlation (r = 0.946, p < 0.01), and MGEs played an important role in the occurrence and evolution of ARGs in water.

We report on the observation of a very low growth velocity of single-walled carbon nanotubes (SWNTs) and consequently the direct length-sorted growth and patterned growth of SWNTs by using a metal-catalyst-free chemical vapor deposition (CVD) process proposed recently by our group, in which SiO2 serves as catalyst. We found that the growth velocity of the SWNTs from SiO2 catalyst is only 8.3 nm/s, which is about 300 times slower than that of the commonly used iron group catalysts (Co as a counterpart catalyst in this study). Such a slow growth velocity renders direct length-sorted growth of SWNTs, especially for short SWNTs with hundreds of nanometers in length. By simply adjusting the growth duration, SWNTs with average lengths of 149, 342, and 483 nm were selectively obtained and SWNTs as short as ∼20 nm in length can be grown directly. Moreover, comparative studies indicate that the SiO2 catalyst possesses a much longer catalytic active time, showing sharp contrast with the commonly used Co catalyst which quickly loses its catalytic activity. Taking advantage of the very slow growth velocity of the SiO2 catalyst, patterned growth of SWNT networks confined in a narrow region of <5 μm was also achieved. The short SWNTs may show intriguing physics owing to their finite length effect and are attractive for various practical applications.

Summary The maternal‐to‐zygotic transition (MZT) is characterized by the turnover of zygote development from maternal to zygotic control, and has been extensively studied in animals. A majority of studies have suggested that early embryogenesis is maternally controlled and that the zygotic genome remains transcriptionally inactive prior to the MZT. However, little is known about the MZT in higher plants, and its timing and impact remain uncharacterized. Here, we constructed cDNA libraries from tobacco ( Nicotiana tabacum ) egg cells, zygotes and two‐celled embryos for gene expression profiling analysis, followed by RT‐PCR confirmation. These analyses, together with experiments using zygote microculture coupled with transcription inhibition, revealed that a marked change in transcript profiles occurs approximately 50 h after fertilization, and that the MZT is initiated prior to zygotic division in tobacco. Although maternal transcripts deposited in egg cells support several early developmental processes, they appear to be insufficient for zygotic polar growth and subsequent cell divisions. Thus, we propose that de novo transcripts are probably required to trigger embryogenesis in later zygotes in tobacco.

Summary Polycomb group ( P c G ) proteins are gene repressors that help to maintain cellular identity during development via chromatin remodeling. Fertilization‐independent endosperm ( FIE ), a member of the P c G complex, operates extensively in plant development, but its role in rice has not been fully investigated to date. We report the isolation and characterization of a P c G member in rice, which was designated O s FIE 2 for O ryza sativa F ertilization‐ I ndependent E ndosperm 2. O s FIE 2 is a single‐copy gene in the rice genome and shows a universal expression pattern. The O s FIE 2 RNA i lines displayed pleiotropic phenotypes in vegetative and reproductive organ generation. In unfertilized lines, endosperm formation could be triggered without embryo formation, which indicates that FIE is indeed involved in the suppression of autonomous endosperm development in rice. Furthermore, lateral root generation was promoted early in the roots of O s FIE 2 RNA i lines, whereas the primary root was premature and highly differentiated. As the root tip stem cell differentiated, QHB , the gene required for stem cell maintenance in the quiescent center, was down‐regulated. Our data suggest that the O s FIE 2– P c G complex is vital for rice reproduction and endosperm formation. Its role in stem cell maintenance suggests that the gene is functionally conserved in plants as well as animals.

Global genomic repair (GGR) and transcription coupled repair (TCR) are two pathways of nucleotide excision repair (NER) that differ in the damage recognition step. How NER factors, especially GGR factors, access DNA damage in the chromatin of eukaryotic cells has been poorly understood. Dot1, a histone methyltransferase required for methylation of histone H3 lysine 79 (H3K79), has been shown to confer yeast cells with resistance to DNA-damaging agents and play a role in activation of DNA damage checkpoints. Here, we show that Dot1 and H3K79 methylation are required for GGR in both nucleosomal core regions and internucleosomal linker DNA, but play no role in TCR. H3K79 trimethylation contributes to but is not absolutely required for GGR, and lower levels of H3K79 methylation (mono- and dimethylation) also promote GGR. Our results also indicate that the roles of Dot1 and H3K79 methylation in GGR are not achieved by either activating DNA damage checkpoints or regulating the expression of the GGR-specific factor Rad16. Rather, the methylated H3K79 may serve as a docking site for the GGR machinery on the chromatin. Our studies identified a novel GGR-specific NER factor and unveiled the critical link between a covalent histone modification and GGR. Global genomic repair (GGR) and transcription coupled repair (TCR) are two pathways of nucleotide excision repair (NER) that differ in the damage recognition step. How NER factors, especially GGR factors, access DNA damage in the chromatin of eukaryotic cells has been poorly understood. Dot1, a histone methyltransferase required for methylation of histone H3 lysine 79 (H3K79), has been shown to confer yeast cells with resistance to DNA-damaging agents and play a role in activation of DNA damage checkpoints. Here, we show that Dot1 and H3K79 methylation are required for GGR in both nucleosomal core regions and internucleosomal linker DNA, but play no role in TCR. H3K79 trimethylation contributes to but is not absolutely required for GGR, and lower levels of H3K79 methylation (mono- and dimethylation) also promote GGR. Our results also indicate that the roles of Dot1 and H3K79 methylation in GGR are not achieved by either activating DNA damage checkpoints or regulating the expression of the GGR-specific factor Rad16. Rather, the methylated H3K79 may serve as a docking site for the GGR machinery on the chromatin. Our studies identified a novel GGR-specific NER factor and unveiled the critical link between a covalent histone modification and GGR.

We consider the problem of scheduling n deteriorating jobs with release dates on a single batching machine. Each job's processing time is an increasing simple linear function of its starting time. The machine can process up to b jobs simultaneously as a batch. The objective is to minimize the maximum completion time, i.e., makespan. For the unbounded model, i.e., b = ∞, we obtain an O(n log n) dynamic programming algorithm. For the bounded model, i.e., b < n, we first show that the problem is binary NP-hard even if there are only two distinct release dates. Then we present O(nb) and O((nb/h)h) algorithms for the case where the job processing order is predetermined in advance and for the case where there are h, h ⩾ 2, distinct deteriorating rates, respectively. Furthermore, we provide a fully polynomial-time approximation scheme for the case where the number of distinct release dates is a constant.

Distorted octahedral T′ phase of MoTe 2 has recently attracted significant interest due to its predicted topological states and novel charge transport properties. Here, we report a nondestructive method for determining the crystal orientation of few‐layer T′‐MoTe 2 flakes by polarized Raman spectroscopy. The experimentally observed Raman modes are assigned to eigenmodes of vibrations predicted by density functional theory calculations. Polarized Raman measurements reveal four distinct types of angle‐dependent intensity variations. From group theory, it can be deduced that the intensity of the B g mode reaches a maximum in the configuration when the polarization vector of the incident light is either parallel or orthogonal to the metal–metal zigzag chain direction. The intensity variation of the B g mode cannot be used to unambiguously determine the crystal orientation. Using electron diffraction analysis, it is demonstrated that the intensity of the A g mode at around 162 cm −1 reaches a maximum when the polarization vector of the incident light is parallel to the metal–metal chain direction in the configuration. Furthermore, a simple method is proposed for identifying crystal orientation in nonpolarized Raman spectroscopy.

In order to achieve a uniform distribution of carbon nanotube (CNT) reinforcement in aluminum (Al) matrix, novel nano/micro-sized hybrid reinforcements with CNT growing on the surface of SiC particle (SiCp) was synthesized by a chemical vapor deposition method. Subsequently, the hybrid reinforcement (defined as SiCp(CNT)) was mixed in the pure Al matrix by merely blending powder, as a result, CNT was well-dispersed in the matrix with the help of micro-sized SiCp as a “vehicle”. With the novel reinforcement, SiCp(CNT)/Al composite exhibited both improved elastic modulus of 93 GPa (35% higher than that of Al matrix) and tensile strength of 202 MPa (74% higher than that of Al matrix), mainly because CNT was dispersed uniformly and achieved intimate interfaces with Al matrix with the help of well dispersed SiCp.

Chemoresistance is a major obstacle in chemotherapy of laryngeal carcinoma.Recently, studies indicate that cancer stem cells are responsible for chemotherapy failure.In addition, microRNAs play important roles in tumor initiation, development and multidrug resistance.In the present study, we found that the expression of microRNA-125a was decreased in laryngeal carcinoma tissues and Hep-2 laryngeal cancer stem cells (Hep-2-CSCs).MicroRNA-125a gain-of-function significantly increased the sensitivity of Hep-2-CSCs to cisplatin in vitro and in vivo.Combination with microRNA-125a mimics can decrease the half maximal inhibitory concentration of Hep-2-CSCs to cisplatin.Mechanically, we found that microRNA-125a reverses cisplatin resistance in Hep-2-CSCs by targeting Hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1).Inhibition of HAX-1 by microRNA-125a significantly promotes the cisplatin-induced apoptosis in Hep-2-CSCs through mitochondrial pathway.In addition, multidrug resistance of Hep-2-CSCs to vincristine, etoposide and doxorubicin was greatly improved after the cells were transfected with microRNA-125a mimics.These dates strongly suggested the promotion of microRNA-125a/HAX-1 axis on chemotherapy of laryngeal carcinoma.

Abstract Accurate design of the 2D metal–semiconductor (M–S) heterostructure via the covalent combination of appropriate metallic and semiconducting materials is urgently needed for fabricating high‐performance nanodevices and enhancing catalytic performance. Hence, the lateral epitaxial growth of M–S Sn x Mo 1− x S 2 /MoS 2 heterostructure is precisely prepared with in situ growth of metallic Sn x Mo 1− x S 2 by doping Sn atoms at semiconductor MoS 2 edge via one‐step chemical vapor deposition. The atomically sharp interface of this heterostructure exhibits clearly distinguished performance based on a series of characterizations. The oxygen evolution photoelectrocatalytic performance of the epitaxial M–S heterostructure is 2.5 times higher than that of pure MoS 2 in microreactor, attributed to the efficient electron–hole separation and rapid charge transfer. This growth method provides a general strategy for fabricating seamless M–S lateral heterostructures by controllable doping heteroatoms. The M–S heterostructures show increased carrier migration rate and eliminated Fermi level pinning effect, contributing to their potential in devices and catalytic system.

The usage of molten salts, e.g., Na2MoO4 and Na2WO4, has shown great success in the growth of two-dimensional (2D) transition metal dichalcogenides (TMDCs) by chemical vapor deposition (CVD). In comparison with the halide salt (i.e., NaCl, NaBr, KI)-assisted growth (Salt 1.0), the molten salt-assisted vapor-liquid-solid (VLS) growth technique (Salt 2.0) has improved the reproducibility, efficiency and scalability of synthesizing 2D TMDCs. However, the growth of large-area MoSe2 and WTe2 is still quite challenging with the use Salt 2.0 technique. In this study, a renewed Salt 2.0 technique using mixed salts (e.g., Na2MoO4-Na2SeO3 and Na2WO4-Na2TeO3) is developed for the enhanced CVD growth of 2D MoSe2 and WTe2 crystals with large grain size and yield. Continuous monolayer MoSe2 film with grain size of 100-250 {\mu}m or isolated flakes up to ~ 450 {\mu}m is grown on a halved 2-inch SiO2/Si wafer. Our study further confirms the synergistic effect of Na+ and SeO32- in the enhanced CVD growth of wafer-scale monolayer MoSe2 film. And thus, the addition of Na2SeO3 and Na2TeO3 into the transition metal salts could be a general strategy for the enhanced CVD growth of many other 2D selenides and tellurides.

In this study, we investigated the effects of hydrothermal temperature on molecular reaction pathways and the plant growth of dissolved organic matters (DOMs) in anaerobic digestion sludge (ADS) via post-hydrothermal treatment (PHT). ADS-derived DOMs mainly comprise lignins/carboxyl-rich alicyclic molecules-like and proteins/aliphatic-like compounds featured with enriched N1-2O6-7 and O8-10 fragments. With the increase in the hydrothermal (HT) temperature, the relative abundance of the compounds with < 7O atoms in DOMs gradually increased in HT-treated samples. Furthermore, based on Fourier transforms ion cyclotron resonance mass spectrometry (FT-ICR-MS)-based reactomics, we found that CHON compounds possessed the highest reactivity, and that reaction of carbonyl (loss of glyoxal (−C2H2O2)) with unique reaction precursors possessed the largest number of high-frequency reactions at different HT temperatures. Consequently, DOM160 contained higher levels of N&O heterocyclic compounds and acetic acid, inhibiting pakchoi growth, and causing an increase in superoxide dismutase activity in the roots. However, DOM200 comprised a higher content of amino acids and humic/fulvic acids, increasing indole-3-acetic acid concentration and H+-ATPase activity in the pakchoi roots, thus exhibiting higher plant activity. Overall, the best HT temperature for ADS dewaterability and the recovery of organic matter was found to be 200 °C. These findings proposed a promising approach for synchronously recycling biostimulants, deep dewatering of ADS via PHT, and further improving the carbon–neutral operation of wastewater treatment plants.

ABSTRACT hupA and hupB encode the α and β subunits of the Escherichia coli histone-like protein HU. Here we show that E. coli hup mutants are sensitive to UV in the rec + sbc + , recBC sbcA , recBC sbcBC , umuDC , recF , and recD backgrounds. However, hupAB mutations do not enhance the UV sensitivity of resolvase-deficient recG ruvA strains. hupAB uvrA and hupAB recG strains are supersensitive to UV. hup mutations enhance the UV sensitivity of ruvA strains to a much lesser extent but enhance that of rus-1 ruvA strains to the same extent as for rus + ruv + strains. Our results suggest that HU plays a role in recombinational DNA repair that is not specifically limited to double-strand break repair or daughter strand gap repair; the lack of HU affects the RecG RusA and RuvABC pathways for Holliday junction processing equally if the two pathways are equally active in recombinational repair; the function of HU is not in the substrate processing step or in the RecFOR-directed synapsis action during recombinational repair. Furthermore, the UV sensitivity of hup mutants cannot be suppressed by overexpression of wild-type or mutant gyrB , which confers novobiocin resistance, or by different concentrations of a gyrase inhibitor that can increase or decrease the supercoiling of chromosomal DNA.

In eukaryotic cells, transcription coupled nucleotide excision repair (TCR) is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the transcribed strand of a gene. Rad26, the yeast homolog of the human Cockayne syndrome group B (CSB) protein, plays an important role in TCR. Spt4, a transcription elongation factor that forms a complex with Spt5, has been shown to suppress TCR in rad26Delta cells. Here we present evidence that Spt4 indirectly suppresses Rad26-independent TCR by protecting Spt5 from degradation and stabilizing the interaction of Spt5 with Pol II. We further found that the C-terminal repeat (CTR) domain of Spt5, which is dispensable for cell viability and is not involved in interactions with Spt4 and Pol II, plays an important role in the suppression. The Spt5 CTR is phosphorylated by the Bur kinase. Inactivation of the Bur kinase partially alleviates TCR in rad26Delta cells. We propose that the Spt5 CTR suppresses Rad26-independent TCR by serving as a platform for assembly of a multiple protein suppressor complex that is associated with Pol II. Phosphorylation of the Spt5 CTR by the Bur kinase may facilitate the assembly of the suppressor complex.

To evaluate the role of 18FDG PET-CT for screening distant metastases before salvage treatment in patients with suspected recurrent head and neck cancer. Studies about 18FDG PET-CT were systematically searched in the MEDLINE and EMBASE databases (last update October 05, 2013). We calculated sensitivities, specificities, positive likelihood ratios and negative likelihood ratios, and constructed summary receiver operating characteristic curves for 18FDG PET-CT. Ten PET-CT studies (756 patients and 797 imaging examinations) were identified. The sensitivity, specificity, positive likelihood ratio and negative likelihood ratio for 18FDG PET-CT were 0.92 (95% CI=0.83-0.96), 0.95 (95% CI=0.91-0.97), 16.7 (95% CI=9.9-28.4), and 0.09 (95% CI=0.04-0.18), respectively. Overall weighted area under the curve was 0.97 (95% CI=0.96-0.98). 18FDG PET-CT has high sensitivity and accuracy for screening distant metastases before salvage treatment in patients with suspected recurrent head and neck cancer.

This paper considers the problem of scheduling deteriorating jobs and due date assignment on a single machine. The actual processing time of a job is a linear increasing function of its starting time. The problem is to determine the optimal due dates and the processing sequence simultaneously to minimize costs for earliness, due date assignment and weighted number of tardy jobs. We present polynomial-time algorithms to solve the problem in the case of two popular due date assignment methods: CON and SLK.

Flowering time is an important agronomic trait that is highly influenced by the environment. To elucidate the genetic mechanism of flowering time in rapeseed (Brassica napus L.), a genome-wide QTL analysis was performed in a doubled haploid population grown in winter, semi-winter and spring ecological conditions. Fifty-five consensus QTLs were identified after combining phenotype and genomic data, including 12 environment-stable QTLs and 43 environment-specific QTLs. Importantly, six major QTLs for flowering time were identified, of which two were considered environment-specific QTLs in spring ecological condition and four were considered environment-stable QTLs in winter and semi-winter ecological conditions. Through QTL comparison, 18 QTLs were colocalized with QTLs from six other published studies. Combining the candidate genes with their functional annotation, in 49 of 55 consensus QTLs, 151 candidate genes in B. napus corresponding to 95 homologous genes in Arabidopsis thaliana related to flowering were identified, including BnaC03g32910D (CO), BnaA02g12130D (FT) and BnaA03g13630D (FLC). Most of the candidate genes were involved in different flowering regulatory pathways. Based on re-sequencing and differences in sequence annotation between the two parents, we found that regions containing some candidate genes have numerous non-frameshift InDels and many non- synonymous mutations, which might directly lead to gene functional variation. Flowering time was negativly correlated with seed yield and thousand seed weight based on a QTL comparison of flowering time and seed yield traits, which has implications in breeding new early-maturing varieties of B. napus. Moreover, a putative flowering regulatory network was constructed, including the photoperiod, circadian clock, vernalization, autonomous and gibberellin pathways. Multiple copies of genes led to functional difference among the different copies of homologous genes, which also increased the complexity of the flowering regulatory networks. Taken together, the present results not only provide new insights into the genetic regulatory network underlying the control of flowering time but also improve our understanding of flowering time regulatory pathways in rapeseed.

Two-dimensional van der Waals heterostructures are attractive candidates for optoelectronic nanodevice applications. The charge transport process in these systems has been extensively investigated, however the effect of coupling between specific electronic states on the charge transfer process is not completely established yet. Here, interfacial charge transfer (CT) in the MoS2/graphene/SiO2 heterostructure is investigated from static and dynamic points of view. Static CT in the MoS2-graphene interface was elucidated by an intensity quenching, broadening and a blueshift of the photoluminescence peaks. Atomic and electronic state-specific CT dynamics on a femtosecond timescale are characterized using a core-hole clock approach and using the S1s core-hole lifetime as an internal clock. We demonstrate that the femtosecond electron transfer pathway in the MoS2/SiO2 heterostructure is mainly due to the electronic coupling between S3p-Mo4d states forming the Mo-S covalent bond in the MoS2 layer. For the MoS2/graphene/SiO2 heterostructure, we identify, with the support of density functional calculations, new pathways that arise due to the high density of empty electronic states of the graphene conduction band. The latter makes the transfer process time in the MoS2/graphene/SiO2/Si twice as fast as in the MoS2/SiO2/Si sample. Our results show that ultrafast electron delocalization pathways in van der Waals heterostructures are dependent on the electronic properties of each involved 2D material, creating opportunities to modulate their transport properties.

Defects are commonly found in two-dimensional (2D) transition-metal dichalcogenide (TMD) materials. Such defects usually dictate the optical and electrical properties of TMDs. It is thus important to develop techniques to characterize the defects directly with good spatial resolution, specificity, and throughput. Herein, we demonstrate that Kelvin probe force microscopy (KPFM) is a versatile technique for this task. It is able to unveil defect heterogeneity of 2D materials with a spatial resolution of 10 nm and energy sensitivity better than 10 meV. KPFM mappings of monolayer WS2 exhibit interesting work function variances that are associated with defects distribution. This finding is verified by aberration-corrected scanning transmission electron microscopy and density functional theory calculations. In particular, a strong correlation among the work function, electrical and optical responses to the defects is revealed. Our findings demonstrate the potential of KPFM as an effective tool for exploring the intrinsic defects in TMDs.

Molecularly thin two-dimensional (2D) semiconductors are emerging as photocatalysts owing to their layer-number-dependent quantum effects and high charge separation efficiency. However, the correlation among the dimensionality, crystallinity, and photocatalytic activity of such 2D nanomaterials remains unclear. Herein, a Ag photoreduction technique coupled with microscopic analyses is employed to spatially resolve the photocatalytic activity of MoS2 as a model catalyst. Interestingly, we find that only monolayer (1L)-MoS2 is active for a Ag photoreduction reaction. The photocatalytic activity of 1L-MoS2 is enhanced by a built-in electrical field originated from the MoS2/SiO2 interface, instead of by the specific surface structure and quantum electronic state of 1L-MoS2. Furthermore, we observe photocatalytic active sites to be geometrically distributed on triangular 1L-MoS2 crystals, wherein the Ag particles are preferentially deposited on the outermost zigzag edges and defective inner parts of the triangular grains. The degradation of photocatalytic activity and electron mobility with the formation of Mo(VI) species indicates that the species inhibit the in-plane diffusion of the photogenerated electrons to the reductive sites. The monolayer-selectivity, activation, and inactivation mechanisms, unveiled in this work, will offer future directions in designing 2D nanophotocatalysts.

Interlayer coupling in two-dimensional (2D) materials with specific stacking modes results in angle-dependent electrical and optical behaviors. Compared to multilayered WS2 with a 0° twist angle grown under normal conditions, WS2 sheets with a 60° twist angle are prepared by chemical vapor deposition (CVD) with the assistance of tin (Sn) for reducing stacking energy. The layer number can be effectively regulated by the growth temperature and hydrogen flow. The 2H-like phase of the 60° bilayer structure is revealed by scanning transmission electron microscopy (STEM), displaying intense photoluminescence (PL) variation and diverse second harmonic generation due to interlayer coupling. The bilayer WS2 with various twist angles can be obtained for angle-dependent Raman/PL response. Density functional theory (DFT) calculations demonstrate that heteroatom Sn can tip the balance between 0° and 60° bilayers, facilitating the formation of the bilayer with a 60° stacking angle. The heteroatom-assisted approach provides a general strategy for the self-assembly of 2D materials. The heterostructures with tunable twist angles can be prepared, which broadens their applications in moiré excitons, spintronics, and valley electronics of transition metal dichalcogenides.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and southeast Asia. Emerging evidence revealed that long noncoding RNAs (lncRNAs) might play important roles in the development and progression of many cancers, including NPC. The functions and mechanisms of the vast majority of lncRNAs involved in NPC remain unknown. In this study, a novel lncRNA RP11-624L4.1 was identified in NPC tissues using next-generation sequencing. <i>In situ</i> hybridization (ISH) was used to analyze the correlation between RP11-624L4.1 expression and the clinicopathological features or prognosis in NPC patients. RNA-Protein Interaction Prediction (RPISeq) predictions and RNA-binding protein immunoprecipitation (RIP) assays were used to identify RP11-624L4.1's interactions with cyclin-dependent kinase 4 (CDK4). As a result, we found that RP11-624L4.1 is hyper-expressed in NPC tissues, which was associated with unfavorable prognosis and clinicopathological features in NPC. By knocking down and overexpressing RP11-624L4.1, we also found that it promotes the proliferation ability of NPC <i>in vitro</i> and <i>in vivo</i> through the CDK4/6-Cyclin D1-Rb-E2F1 pathway. Overexpression of CDK4 in knocking down RP11-624L4.1 cells can partially rescue NPC promotion, indicating its role in the RP11-624L4.1-CDK4/6-Cyclin D1-Rb-E2F1 pathway. Taken together, RP11-624L4.1 is required for NPC unfavorable prognosis and proliferation through the CDK4/6-Cyclin D1-Rb-E2F1 pathway, which may be a novel therapeutic target and prognostic in patients with NPC.

Two-dimensional transition-metal dichalcogenide monolayers have remarkably large optical nonlinearity. However, the nonlinear optical conversion efficiency in monolayer transition-metal dichalcogenides is typically low due to small light–matter interaction length at the atomic thickness, which significantly obstructs their applications. Here, for the first time, we report broadband (up to ∼150 nm) enhancement of optical nonlinearity in monolayer MoS2 with plasmonic structures. Substantial enhancement of four-wave mixing is demonstrated with the enhancement factor up to three orders of magnitude for broadband frequency conversion, covering the major visible spectral region. The equivalent third-order nonlinearity of the hybrid MoS2-plasmonic structure is in the order of 10–17 m2/V2, far superior (∼10–100-times larger) to the widely used conventional bulk materials (e.g., LiNbO3, BBO) and nanomaterials (e.g., gold nanofilms). Such a considerable and broadband enhancement arises from the strongly confined electric field in the plasmonic structure, promising for numerous nonlinear photonic applications of two-dimensional materials.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the nasopharynx mainly characterized by geographic distribution and EBV infection. Metabolic reprogramming, one of the cancer hallmarks, has been frequently reported in NPCs to adapt to internal energy demands and external environmental pressures. Inevitably, the metabolic reprogramming within the tumor cell will lead to a decreased pH value and diverse nutritional supplements in the tumor-infiltrating micro-environment incorporating immune cells, fibroblasts, and endothelial cells. Accumulated evidence indicates that metabolic reprogramming derived from NPC cells may facilitate cancer progression and immunosuppression by cell-cell communications with their surrounding immune cells. This review presents the dysregulated metabolism processes, including glucose, fatty acid, amino acid, nucleotide metabolism, and their mutual interactions in NPC. Moreover, the potential connections between reprogrammed metabolism, tumor immunity, and associated therapy would be discussed in this review. Accordingly, the development of targets on the interactions between metabolic reprogramming and immune cells may provide assistances to overcome the current treatment resistance in NPC patients.

The CoNiW alloy coating is electrodeposited on the 1045 steel as piston ring materials by electrodeposition method to greatly improve the wear resistance performance. The electrodeposition of CoNiW belongs to a kind of induced co-deposition. The tungstate ions in the plating solution are beneficial to increase the deposition mass. Moreover, tungsten atoms could occupy the lattice junction of nickel and cobalt atoms to generate lattice distortion, which will reduce the grain size, decrease the surface roughness and improve the wear resistance performance. Compared with Co coating and CoNi coating, the CoNiW electrodeposited coating has the best wear resistance performance with the smallest surface roughness (0.179 µm) and the thinnest wear scar (7.4 µm average depth).

Two-dimensional (2D) semiconductors have attracted great attention to extend Moore’s law, which motivates the quest for fast growth of high-quality materials. However, taking MoS2 as an example, current methods yield 2D MoS2 with low growth rate and poor quality with vacancy concentrations three to five orders of magnitude higher than silicon and other commercial semiconductors. Here, we develop a strategy of using intermediate product of iodine as a transport agent to carry metal precursors efficiently for ultrafast growth of high-quality MoS2. The grown MoS2 has the lowest density of sulfur vacancies (~ 1.41 × 1012 cm-2) reported so far and excellent electrical properties with high on/off current ratios of 108 and carrier mobility of 175 cm2V-1s-1. Theoretical calculations show that by incorporating iodine, the nucleation barrier of MoS2 growth with sulfur-terminated edges reduces dramatically. The sufficient supply of precursor and low nucleation energy together boost the ultrafast growth of sub-millimeter MoS2 domains within seconds. This work provides an effective method for ultrafast growth of 2D semiconductors with high quality, which will promote their applications.

We wished to determine where transcription enhanced nucleotide excision repair begins and ends for a Saccharomyces cerevisiae gene transcribed by RNA polymerase II, and to examine the role of the RAD16 gene in repairing upstream, non-transcribed control sequences of such a gene. To do so, we developed a method to study the repair of UV induced cyclobutane pyrimidine dimers (CPDs) at the level of the nucleotide in the control and coding sequences of the MFA2 gene. This gene is active in haploid a mating type cells but inactive in α cells: its regulation is mediated by changes in chromatin structure. DNA from UV irradiated cells was cut with a CPD-specific endonuclease, restricted and selected strands of the MFA2 gene separated from genomic DNA prior to end-labelling and resolution on a sequencing gel. We confirmed repair trends seen using Southern blotting to examine kilobase size fragments, but were additionally able to elucidate subtle differences in repairing portions of the transcribed strand (TS) of MFA2. Enhanced repair of the TS when the gene is active, began well before the start of transcription. Clearly, enhanced repair in this region cannot be due to mRNA synthesis. The repair of CPDs is even further enhanced in the transcribed portion of the TS, and returns to a basal level after the termination of transcription. The approach also revealed that RAD16 has a role in the repair of the TS when MFA2 is active. Removal of CPDs from the TS control region was impaired but not totally defective in a rad16 a mutant. Repair from the TS coding sequence also has a Rad16 component, but a lesser one than for the upstream control sequences, and this was more marked for the sequences towards the end of the transcribed region. The system developed permits further dissection of the relationships between DNA repair, chromatin structure and transcription at the MFA2 locus.

We consider several parallel-machine scheduling problems in which the processing time of a job is a (simple) linear increasing function of its starting time and jobs can be rejected by paying penalties. The objective is to minimize the scheduling cost of the accepted jobs plus the total penalty of the rejected jobs. Three variations of the scheduling cost are considered in this paper. The first is the makespan, the second is the total weighted completion time (for simple linear deterioration), and the third is the total completion time. For the former two problems, we propose two fully polynomial-time approximation schemes to solve them when the number of machines is fixed. For the last problem, we present an optimal O(n2)-time dynamic programming algorithm when the deteriorating rates are equal for all jobs.

We consider a single-machine scheduling problem involving both the due date assignment and job scheduling under a group technology environment. The jobs (orders) of customers are classified into groups according to their production similarities in advance. To achieve production efficiency and save time/money resource, all jobs of the same group are required to be processed contiguously on the machine. A sequence-independent setup time precedes the processing of each group. The due dates are assignable according to one of the following three due date assignment methods: FML-CON, FML-SLK and DIF, where FML-CON means that all jobs within the same group are assigned a common due date, FML-SLK means that all jobs within the same group are assigned an equal flow allowance, and DIF means that each job can be assigned a different due date with no restrictions. The goal is to determine an optimal combination of the due date assignment strategy and job schedule so as to minimize an objective function that includes earliness, tardiness, due date assignment and flow time costs. An O(nlogn) time unified optimization algorithm is provided for all of the above three due date assignment methods.

Transcription coupled nucleotide excision repair (TC-NER) is believed to be triggered by an RNA polymerase stalled at a lesion in the transcribed strand of actively transcribed genes. Rad26, a DNA-dependent ATPase in the family of SWI2/SNF2 chromatin remodeling proteins, plays an important role in TC-NER in Saccharomyces cerevisiae. However, Rad26 is not solely responsible for TC-NER and Rpb9, a nonessential subunit of RNA polymerase II (RNAP II), is largely responsible for Rad26-independent TC-NER. The Rad26-dependent and Rpb9-dependent TC-NER have different efficiencies in genes with different transcription levels and in different regions of a gene. Rad26 becomes entirely or partially dispensable for TC-NER in the absence of Rpb4, another nonessential subunit of RNAP II, or a number of transcription elongation factors (Spt4, Spt5 and the RNAP II associated factor complex). Rad26 may not be a true transcription-repair coupling factor that recruits the repair machinery to the damaged sites where RNAP II stalls. Rather, Rad26 may facilitate TC-NER indirectly, by antagonizing the action of TC-NER repressors that normally promote transcription elongation. The underlying mechanism of how Rad26 functions in TC-NER remains to be elucidated.

The high diffusion rate of sulfur with respect to metal oxide creates precursors that deviate from the stoichiometric ratio, leading to poor growth controllability and defects in the as-grown transition metal dichalcogenides (TMDCs). The introduction of a sulfur precursor with a high melting point is a hopeful strategy to solve these problems. Here, we first introduce sodium sulfate (Na2SO4) as a sulfur precursor, which plays roles in tuning diffusion of source precursors and balancing their mass flux based on the temperature-confined decomposition of Na2SO4. We deduced the specific growth process by characterizing the composition of intermediates; the results show that emissions of sulfur and metal sources were synchronously released and spanning the entire growth stage. This temperature-controlled source-feeding system reduced the diffusion gap between sulfur and metal, which promoted a faster kinetics for reactions. Moreover, this method has the wide applicability for producing other TMDCs.

Abnormal anti-pyramid MoS₂/WS₂ vertical heterostructures were synthesized by a facile single-step chemical vapour deposition.

High-harmonic generation (HHG), an extreme nonlinear optical phenomenon beyond the perturbation regime, is of great significance for various potential applications, such as high-energy ultrashort pulse generation with outstanding spatiotemporal coherence. However, efficient active control of HHG is still challenging due to the weak light-matter interaction displayed by currently known materials. Here, we demonstrate optically controlled HHG in monolayer semiconductors via the engineering of interband polarization. We find that HHG can be efficiently controlled in the excitonic spectral region with modulation depths up to 95% and ultrafast response speeds of several picoseconds. Quantitative time-domain theory of the nonlinear optical susceptibilities in monolayer semiconductors further corroborates these experimental observations. Our demonstration not only offers an in-depth understanding of HHG but also provides an effective approach toward active optical devices for strong-field physics and extreme nonlinear optics.

Transcription-coupled repair (TCR) and global genomic repair (GGR) of UV-induced cyclobutane pyrimidine dimers were investigated in the yeast GAL1-10 genes.Both Rpb9-and Rad26-mediated TCR are confined to the transcribed strands, initiating at upstream sites ϳ100 nucleotides from the upstream activating sequence shared by the two genes.However, TCR initiation sites do not correlate with either transcription start sites or TATA boxes.Rad16-mediated GGR tightly correlates with nucleosome positioning when the genes are repressed and are slow in the nucleosome core and fast in linker DNA.Induction of transcription enhanced GGR in nucleosome core DNA, especially in the nucleosomes around and upstream of the transcription start sites.Furthermore, when the genes were induced, GGR was slower in the transcribed regions than in the upstream regions.Finally, simultaneous deletion of RAD16, RAD26, and RPB9 resulted in no detectable repair in all sites along the region analyzed.Our results suggest that (a) TCR may be initiated by a transcription activator, presumably through the loading of RNA polymerase II, rather than by transcription initiation or elongation per se; (b) TCR and nucleosome disruptionenhanced GGR are the major causes of rapid repair in regions around and upstream of transcription start sites; (c) transcription machinery may hinder access of NER factors to a DNA lesion in the absence of a transcription-repair coupling factor; and (d) other than GGR mediated by Rad16 and TCR mediated by Rad26 and Rpb9, no other nucleotide excision repair pathway exists in these RNA polymerase II-transcribed genes.

A survey of crude plant extracts for DNA polymerase β inhibitors resulted in the identification of a methyl ethyl ketone extract prepared from Knema elegans that strongly inhibited the enzyme. Subsequent bioassay-guided fractionation of the extract, using an assay to monitor the activity of DNA polymerase β, led to the isolation of two potent inhibitors, (+)-myristinins A (1) and D (2), which are known flavans having unusual structures. (+)-Myristinins A and D exhibited IC50 values of 12 and 4.3 μM, respectively, as inhibitors of DNA polymerase β in the presence of bovine serum albumin (BSA), and 2.7 and 1.2 μM in the absence of BSA. As such, they are the most potent DNA polymerase β inhibitors reported to date. Compounds 1 and 2 potentiated the cytotoxicity of bleomycin toward cultured P388D1 cells, reducing the number of viable cells by at least 30% when employed at 9 μM concentration for 6 h in the presence of an otherwise nontoxic concentration of bleomycin (75 nM). Principles 1 and 2 also induced strong Cu2+-dependent DNA strand scission in a DNA cleavage assay. Accordingly, 1 and 2 exhibit two activities, namely, DNA polymerase β inhibition and DNA damage.

We demonstrate that manganese (Mn) can catalyze the growth of single-walled carbon nanotubes (SWNTs) with high efficiency via a chemical vapor deposition process. Dense and uniform SWNT films with high quality were obtained by using a Mn catalyst, as characterized by scanning electron microscopy, Raman spectroscopy, atomic force microscopy, and transmission electron microscopy. Moreover, we found that the surface property of the substrate plays a critical role in the growth efficiency of SWNTs. By deposition of a thin oxide layer (SiO2 or Al2O3) on the top of a SiO2/Si substrate, the growth efficiency of SWNTs was dramatically improved. The successful growth of SWNTs by Mn catalyst provides new experimental information for understanding the growth mechanism of SWNTs, which may be helpful for their controllable synthesis.

Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER) is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4) or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

The effect of sulfur on the catalytic nucleation and growth of single-wall carbon nanotubes (SWCNTs) from an iron catalyst was investigated in situ by transmission electron microscopy (TEM). The catalyst precursor of ferrocene and growth promoter of sulfur were selectively loaded inside of the hollow core of multiwall CNTs with open ends, which served as a nanoreactor powered by applying a voltage inside of the chamber of a TEM. It was found that a SWCNT nucleated and grew perpendicularly from a region of the catalyst nanoparticle surface, instead of the normal tangential growth that occurs with no sulfur addition. Our in situ TEM observation combined with CVD growth studies suggests that sulfur functions to promote the nucleation and growth of SWCNTs by forming inhomogeneous local active sites and modifying the interface bonding between catalysts and precipitated graphitic layers, so that carbon caps can be lifted off from the catalyst particle.

Selective removal of metallic single-walled carbon nanotubes (SWCNTs) and consequent enrichment of semiconducting SWCNTs were achieved through an efficient carbothermic reaction with a NiO thin film at a relatively low temperature of 350 °C. All-SWCNT field effect transistors (FETs) were fabricated with the aid of a patterned NiO mask, in which the as-grown SWCNTs behaving as source/drain electrodes and the remaining semiconducting SWCNTs that survive in the carbothermic reaction as a channel material. The all-SWCNT FETs demonstrate improved current ON/OFF ratios of ∼103.

Rad26, a DNA dependent ATPase that is homologous to human CSB, has been well known to play an important role in transcription coupled DNA repair (TCR) in the yeast Saccharomyces cerevisiae Sen1, a DNA/RNA helicase that is essential for yeast cell viability and homologous to human senataxin, has been known to be required for transcriptional termination of short noncoding RNA genes and for a fail-safe transcriptional termination mechanism of protein-coding genes. Sen1 has also been shown to protect the yeast genome from transcription-associated recombination by resolving RNA:DNA hybrids naturally formed during transcription. Here, we show that the N-terminal non-essential region of Sen1 plays an important role in TCR, whereas the C-terminal nonessential region and the helicase activity of Sen1 are largely dispensable for the repair. Unlike Rad26, which becomes completely dispensable for TCR in cells lacking the TCR repressor Spt4, Sen1 is still required for efficient TCR in the absence of Spt4. Also unlike Rad26, which is important for repair at many but not all damaged sites in the transcribed strand of a gene, Sen1 is required for efficient repair at essentially all the damaged sites. Our results indicate that Sen1 plays a more direct role than Rad26 in TCR.

Bisphenol A (BPA) can be accumulated into the human body via food intake and inhalation. Numerous studies indicated that BPA can trigger the tumorigenesis and progression of cancer cells. Laryngeal cancer cells can be exposed to BPA directly via food digestion, while there were very limited data concerning the effect of BPA on the development of laryngeal squamous cell carcinoma (LSCC). Our present study revealed that nanomolar BPA can trigger the proliferation of LSCC cells. Bisphenol A also increased the in vitro migration and invasion of LSCC cells and upregulated the expression of matrix metallopeptidase 2. Among various chemokines tested, the expression of IL-6 was significantly increased in LSCC cells treated with BPA for 24 hours. Neutralization antibody of IL-6 or si-IL-6 can attenuate BPA-induced proliferation and migration of LSCC cells. Targeted inhibition of G protein-coupled estrogen receptor, while not estrogen receptor (ERα), abolished BPA-induced IL-6 expression, proliferation, and migration of LSCC cells. The increased IL-6 can further activate its downstream signal molecule STAT3, which was evidenced by the results of increased phosphorylation and nuclear translocation of STAT3, while si-IL-6 and si-GPER can both reverse BPA-induced activation of STAT3. Collectively, our present study revealed that BPA can trigger the progression of LSCC via GPER-mediated upregulation of IL-6. Therefore, more attention should be paid for the BPA exposure on the development of laryngeal cancer.

Antibiotic resistance genes (ARGs) are considered to be emerging pollutants related to human activities. The rapid development of global urbanization has expanded human activities, thereby exacerbating the global human health risks caused by antibiotic resistance genes. The effects of urban and rural environments are multifarious, which makes the source and distribution of ARGs in the environment diversification. Understanding the distribution and spread of ARGs is essential for studying the environmental behavior of ARGs. In this study, the occurrence 296 genes were detected by the high-throughput qPCR technology, and FC value was used to analyze the diversity of ARGs and mobile genetic elements (MGEs) in sediments between urban and rural areas of the Liaohe River Basin, China. The co-occurrence of MGEs and ARGs was analyzed using network to decipher core genes. A total of 187 ARGs and 10 MGEs were detected in all sediment samples. The average number of genes detected in urban sites is 89 higher than that in rural sites. The high abundance and various types of ARGs and MGEs detected in urban river sediments indicate that the occurrence of urban ARGs is more complex. MGEs were detected high levels and were significantly correlated with the abundance and diversity of ARGs in river sediments providing evidence that MGEs were related to the occurrence and distribution of ARGs and tnpA (tnpA-07, tnpA-01, and tnpA-03) gene were at the key position of co-occurrence of various types of ARGs.

This review aims to elucidate relevant challenging issues on controllable wafer-scale preparation, additive patterning, and heterogeneous integration of van der Waals single crystals with uniform morphology and crystallographic orientation.

Phase transformation lies at the heart of materials science because it allows for the control of structural phases of solids with desired properties. It has long been a challenge to manipulate phase transformations in crystals at the nanoscale with designed interfaces and compositions. Here in situ electron microscopy is employed to fabricate novel 2D phases with different stoichiometries in monolayer MoS2 and MoSe2 . The multiphase transformations: MoS2 → Mo4 S6 and MoSe2 → Mo6 Se6 which are highly localized with atomically sharp boundaries are observed. Their atomic mechanisms are determined as chalcogen 2H ↔ 1T sliding, cation shift, and commensurate lattice reconstructions, resulting in decreasing direct bandgaps and even a semiconductor-metal transition. These results will be a paradigm for the manipulation of multiphase heterostructures with controlled compositions and sharp interfaces, which will guide the future phase engineered electronics and optoelectronics of metal chalcogenides.

Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules, such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here, we report an approach to control the number of bound RTX molecules (0–4) in functional rat TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.70 to 1.86 kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX. Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules, such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here, we report an approach to control the number of bound RTX molecules (0–4) in functional rat TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.70 to 1.86 kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX. Allosteric coupling links structurally separate function domains together through a global conformational rearrangement, which, in protein complexes, introduces cooperativity (1Monod J. Jacob F. Teleonomic mechanisms in cellular metabolism, growth, and differentiation.Cold Spring Harb. Symp. Quant. Biol. 1961; 26: 389-401Crossref PubMed Google Scholar). Cooperative activation can substantially enhance the sensitivity to a stimulus, hence bestows a significant functional advantage to multisubunit complexes. Indeed, multisubunit complex formation is a common phenomenon in biology (2Hill A.V. Paganini-Hill A. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves.J. Physiol. 1910; 40: 4-7Google Scholar). Even though diverse models have been satisfactorily used to describe the overall cooperative behavior in protein complexes (3Monod J. Wyman J. Changeux J.P. On the nature of allosteric transitions: a plausible model.J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Google Scholar, 4Koshland Jr., D.E. Némethy G. Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits.Biochemistry. 1966; 5: 365-385Crossref PubMed Google Scholar), it remains a major challenge to directly access cooperative activation among subunits as it occurs, limiting our understanding of the underlying mechanism. The Monod–Wyman–Changeux (MWC) model proposed over half a century ago (3Monod J. Wyman J. Changeux J.P. On the nature of allosteric transitions: a plausible model.J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Google Scholar) postulates that ligand binding to individual subunits promotes a concerted activation transition by an identical cooperative factor, f, which reflects energetic contribution by each subunit (Fig. 1A). However, exactly because of cooperative activation, the intermediate states (shaded in Fig. 1A) are rapidly traversed; their energetic contributions are rarely determined individually. Reflecting this challenge, a common practice has been to fit observed overall activities to a Hill function, yielding a slope factor estimate that is neither the cooperative factor nor the number of subunits but their combined contribution (5Weiss J.N. The hill equation revisited: uses and misuses.FASEB J. 1997; 11: 835-841Crossref PubMed Google Scholar). Biological ion channels are usually made of multiple functionally coupled subunits or repeating domains (6Hille B. Ion channels of Excitable Membranes.3rd Ed. Sinauer Associates Inc, Sunderland, MA2001Google Scholar, 7Zheng J. Trudeau M.C. Handbook of Ion Channels. Crc Press, Boca Raton, FL2015Crossref Google Scholar). The capsaicin (CAP) receptor TRPV1 is a representative multisubunit ion channel (8Caterina M.J. et al.The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (7177) Google Scholar, 9Cao E. Liao M. Cheng Y. Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms.Nature. 2013; 504: 113-118Crossref PubMed Scopus (754) Google Scholar) whose activation exhibits allosteric properties (10Latorre R. Brauchi S. Orta G. Zaelzer C. Vargas G. ThermoTRP channels as modular proteins with allosteric gating.Cell Calcium. 2007; 42: 427-438Crossref PubMed Scopus (167) Google Scholar, 11Matta J.A. Ahern G.P. Voltage is a partial activator of rat thermosensitive TRP channels.J. Physiol. 2007; 585: 469-482Crossref PubMed Scopus (155) Google Scholar, 12Jara-Oseguera A. Islas L.D. The role of allosteric coupling on thermal activation of thermo-TRP channels.Biophys. J. 2013; 104: 2160-2169Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 13Cao X. Ma L. Yang F. Wang K. Zheng J. Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold.J. Gen. Physiol. 2014; 143: 75-90Crossref PubMed Scopus (34) Google Scholar). TRPV1 is known to serve as a polymodal nociceptor; its activation by vanilloids has been investigated in both functional and structural studies (14Yang F. Zheng J. Understand spiciness: mechanism of TRPV1 channel activation by capsaicin.Protein Cell. 2017; 8: 169-177Crossref PubMed Scopus (184) Google Scholar). Vanilloid compounds occupy a binding pocket within the transmembrane region surrounded by multiple transmembrane segments and the S4–S5 linker (9Cao E. Liao M. Cheng Y. Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms.Nature. 2013; 504: 113-118Crossref PubMed Scopus (754) Google Scholar). In a typical recording of the mouse TRPV1 activated by CAP, current amplitude reached over 90% maximum within hundreds of milliseconds (Fig. S1). Even though this activation process is slow among ion channels, the intermediate states were swiftly traversed and could not be individually identified and studied (15Hui K. Liu B. Qin F. Capsaicin activation of the pain receptor, VR1: multiple open states from both partial and full binding.Biophys. J. 2003; 84: 2957-2968Abstract Full Text Full Text PDF PubMed Google Scholar). Methods to slow down activation and to fix gating transition in individual subunits were developed in this study, which allowed direct assessment of the property of intermediate states and subunit coupling at equilibrium under physiological conditions. We first used resiniferatoxin (RTX), a potent TRPV1 activator (8Caterina M.J. et al.The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (7177) Google Scholar), to replace CAP as the agonist. RTX binds in the same vanilloid-binding pocket as CAP (9Cao E. Liao M. Cheng Y. Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms.Nature. 2013; 504: 113-118Crossref PubMed Scopus (754) Google Scholar) but activates the channel with an extremely slow (in minutes) time course; RTX-induced activation is also irreversible (Fig. 1B). The slow activation time course revealed a clear transition phase of increasing open probability (Po) in single-channel recordings (Fig. 1, C and F), indicating sojourns in unstable intermediate open states as suggested by the MWC model. However, these transient and stochastic events were observed under nonequilibrium conditions, hence were still difficult to analyze. From the RTX–TRPV1 complex structures (9Cao E. Liao M. Cheng Y. Julius D. TRPV1 structures in distinct conformations reveal activation mechanisms.Nature. 2013; 504: 113-118Crossref PubMed Scopus (754) Google Scholar, 16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), we identified key channel residues that directly interact with a bound RTX (Fig. S2). Mutations to these residues in most cases either exerted minor gating effects or eliminated function (Fig. S3). Mutating Y512 to alanine retained the slow activation property of the wildtype channel but noticeably made RTX activation completely reversible (Fig. 1D). We have previously suggested that Y512 serves to physically block a bound vanilloid molecule from exiting the binding pocket (17Yang F. et al.Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.Nat. Chem. Biol. 2015; 11: 518-524Crossref PubMed Scopus (200) Google Scholar), in agreement with structural (16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and functional (18Hazan A. Basu A. Zalcman N. Matzner H. Priel A. Tyrosine residue in the TRPV1 vanilloid binding pocket regulates deactivation kinetics.J. Biol. Chem. 2016; 291: 13855-13863Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) observations. Molecular docking of RTX to TRPV1 structures in the closed (C1 and C2) and open (O1) states using RosettaLigand (RosettaCommons, https://www.rosettacommons.org/software/license-and-download) (19DeLuca S. Khar K. Meiler J. Fully flexible docking of medium sized ligand libraries with rosettaligand.PLoS One. 2015; 10e0132508Crossref Scopus (69) Google Scholar, 20Brown B.P. et al.Introduction to the BioChemical Library (BCL): an application-Based open-source toolkit for integrated cheminformatics and machine learning in computer-aided drug discovery.Front. Pharmacol. 2022; 13833099Crossref Scopus (3) Google Scholar) suggested reductions in Rosetta binding energy by this point mutation, in supportive of weakening of RTX binding (Fig. S4). After washing off RTX, Y512A channels could be activated by 2-APB, CAP, and RTX similar to naïve channels without detectable desensitization (under the Ca2+-free condition). Single-channel recordings confirmed that Y512A traversed the transition phase during both activation and deactivation time courses (Figs. 1, E and G and S5). Taking advantage of the reversibility property of the Y512A mutant, we studied rat TRPV1 concatemers containing different numbers of wildtype and Y511A mutant (equivalent to Y512A in mouse TRPV1) subunits (Fig. 2A). These concatemer channels were previously shown to respond to CAP in a concentration-dependent manner, with the CAP potency progressively decreased as the number of mutant protomers increased (21Hazan A. Kumar R. Matzner H. Priel A. The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.Sci. Rep. 2015; 5: 1-13Crossref Scopus (37) Google Scholar). We confirmed that the spontaneous Po of each concatemer matched that of wildtype channels (Fig. 2, D and E). We observed with macroscopic (Fig. 2B) and single-channel recordings (Fig. S6) that RTX could fully activate all concatemer channels. Importantly, currents from these concatemers exhibited gradually enhanced reversibility upon washing off RTX, with channels containing all or three wildtype subunits (YYYY and YYYA, respectively) exhibiting little current deactivation, whereas those with all Y511A subunits (AAAA) deactivated completely like channels formed by unlinked monomeric mouse Y512A subunits (Fig. 1D). We further confirmed that the remaining currents were not because of an unstable patch (Fig. 2B) but instead represented steady intermediate Pos (Figs. 2C and S6). These behaviors are expected as RTX binding to the Y511A subunits in concatemers is reversible but irreversible when bound to the wildtype subunits. Therefore, after extended washing, the concatemer channels contained a fixed number (zero to four) of bound RTX molecules. The concatemers allowed us to characterize each vertical equilibrium of the MWC model (Fig. 1A) in isolation. For example, after fully activating the YYAA channels and washing off RTX from the mutant subunits, each channel contained two RTX molecules (the third column of the model). The Po value of these channels could be determined from both macroscopic currents (77 ± 3% of the maximum response; Fig. 2D) and single-channel currents (62 ± 10%; Fig. 2E) at the steady state. Po estimates for YYYY and AAAA channels matched those from channels made of wildtype and mutant monomeric subunits, respectively (Fig. 2, D and E). The Po measurements from all concatemers could be satisfactorily fitted to a general formula derived from the MWC model that describes the relationship between Po and the number of bound ligands (Fig. 2F). Transformation of the formula yields a linear function that allows easy estimation of the equilibrium constant of unliganded channels, L0, and the cooperative factor, f. Figure 2G shows results from the single-channel data. We estimated the L0 value to be 0.007, equivalent to a resting Po of 0.7%, which is consistent with experimental observations from wildtype channels (22Yang F. et al.The conformational wave in capsaicin activation of transient receptor potential vanilloid 1 ion channel.Nat. Commun. 2018; 9: 1-9PubMed Google Scholar); we estimated the f value to be 23.1, equivalent to an activation energy of 1.86 kcal/mol per RTX binding (Fig. 2G). It is noticed that a linear fit agreed with experimental measurements remarkably well, suggesting that subunit contributions to activation are indeed equal, as postulated by the MWC theorem. The position of wildtype and mutant subunits within the concatemers had no obvious influence on the activation energy, as data from concatemers YAYA and AYAY agreed reasonably well with that from YYAA (Figs. 2G and S7), an observation consistent with the new cryo-EM data showing that RTX binding has no position preference (16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Where does the 1.86 kcal/mol activation energy arise from? For the isolated transition shown in Figure 2F, the equilibrium can be altered at either the closed state or the open state, both of which could contribute to an increased equilibrium constant. To quantify energetic effects of RTX binding to the closed and open states, we analyzed single-channel dwell-time distributions of the concatemers at steady state (Fig. 3). When results from all concatemers were compared, we found that the mean dwell time of the closed state decreased exponentially with an increasing number of bound RTX, whereas the mean dwell time of the open state increased exponentially (Fig. 3B). Equilibrium constant values estimated from dwell-time measurements matched closely with those estimated from Po measurements (Fig. 3C). From linear fittings of the log-transformed dwell times (Fig. 3B), we estimated that each RTX binding destabilized the closed state by 1.17 kcal/mol, stabilized the open state by 0.53 kcal/mol, therefore producing a total of 1.70 kcal/mol of activation energy. Therefore, activation of TRPV1 by RTX originates predominately from destabilization of the closed state. Measurements from the concatemers revealed that the Po values with 0 to 4 bound RTX molecules. Using this information, we could tentatively identify the intermediate open states produced by sequential bindings of RTX during the course of activation. A typical time course of the wildtype channel single-channel activation exhibited distinct open states with low, moderate, and high Po values, corresponding to one-, two-, and three- or four-bound RTX molecules, respectively (Fig. 4A). All these open states, observed in the same single-channel recording, exhibited identical amplitudes (Fig. 4, B and C). Similar observations were made from the concatemers. This finding is consistent with the classic MWC model in which the multisubunit complex exists in two (tense and relaxed) conformations, with ligand binding progressively shifting the equilibrium toward the relaxed, a.k.a. open, conformation (3Monod J. Wyman J. Changeux J.P. On the nature of allosteric transitions: a plausible model.J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Google Scholar). Our results suggest that TRPV1 activated by RTX has a single open pore conformation. Previously, we reported that structurally related natural vanilloids from chili peppers and gingers as well as synthetic CAP derivatives induce similar conformational changes of the vanilloid-binding pocket of TRPV1, even though they may bind in different poses (23Vu S. Singh V. Wulff H. Yarov-Yarovoy V. Zheng J. New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket.Elife. 2020; 9Crossref Scopus (7) Google Scholar, 24Yin Y. et al.Structural mechanisms underlying activation of TRPV1 channels by pungent compounds in gingers.Br. J. Pharmacol. 2019; 176: 3364-3377PubMed Google Scholar, 25Dong Y. et al.A distinct structural mechanism underlies TRPV1 activation by piperine.Biochem. Biophysical Res. Commun. 2019; 516: 365-372Crossref PubMed Scopus (26) Google Scholar). We now present data suggesting that the activated channel pore adopts the same open conformation when a varying number of vanilloid-binding pockets are occupied by RTX, consistent of pore opening being a concerted transition. Putting together, these observations support the general assumption that TRPV1 behaves as an allosteric protein (10Latorre R. Brauchi S. Orta G. Zaelzer C. Vargas G. ThermoTRP channels as modular proteins with allosteric gating.Cell Calcium. 2007; 42: 427-438Crossref PubMed Scopus (167) Google Scholar, 11Matta J.A. Ahern G.P. Voltage is a partial activator of rat thermosensitive TRP channels.J. Physiol. 2007; 585: 469-482Crossref PubMed Scopus (155) Google Scholar, 12Jara-Oseguera A. Islas L.D. The role of allosteric coupling on thermal activation of thermo-TRP channels.Biophys. J. 2013; 104: 2160-2169Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 13Cao X. Ma L. Yang F. Wang K. Zheng J. Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold.J. Gen. Physiol. 2014; 143: 75-90Crossref PubMed Scopus (34) Google Scholar). By fixing the number of bound RTX molecules, we were able to directly quantify equilibrium properties from otherwise unstable intermediate states at both macroscopic and single-molecule resolutions. We observed that binding of RTX to each of the four subunits yielded an equal activation energy, as predicted by the classic MWC model for homotropic cooperativity. A recent structural study of RTX–TRPV1 complexes indeed suggested nonpreference in RTX binding to the four subunits (16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The activation energy, estimated at 1.70 to 1.86 kcal/mol per liganded subunit, is comparable to that of other channels such as CNG channels (0.84 to 1.3 kcal/mol) determined indirectly from overall ligand activation behaviors (26Schirmeyer J. et al.Thermodynamic profile of mutual subunit control in a heteromeric receptor.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2100469118Crossref PubMed Scopus (4) Google Scholar, 27Gordon S.E. Zagotta W.N. Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels.Neuron. 1995; 14: 857-864Abstract Full Text PDF PubMed Scopus (142) Google Scholar). The comparable maximal Po of CAP-activated channels indicates that CAP binding is expected to produce a similar activation energy (17Yang F. et al.Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.Nat. Chem. Biol. 2015; 11: 518-524Crossref PubMed Scopus (200) Google Scholar). Combining the activation energies from multiple subunits contributes to an exponential increase in Po (the 1.86 × 4 = 7.44 kcal/mol activation energy shifts the closed-to-open equilibrium toward the open state by over 280,000 folds) (Fig. 4D). For TRPV1 activated by RTX, the shift in equilibrium originates mostly from destabilization of the closed state. Energetic contributions from the four subunits represent substantial activation cooperativity in TRPV1. Indeed, cooperative activation is a common phenomenon for diverse allosteric proteins. However, a general challenge for studying a cooperative process is to capture the intermediate states with either functional methods or structural methods. In the absence of direct information concerning the intermediate states, a mechanistic interpretation is often speculative. The MWC model for allosteric proteins made of identical subunits (3Monod J. Wyman J. Changeux J.P. On the nature of allosteric transitions: a plausible model.J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Google Scholar) has been successfully applied to a wide variety of proteins including those made of different subunits. Nonetheless, equivalence in subunit contributions, a key feature distinguishing it from a Koshland–Némethy–Filmer–type sequential model (4Koshland Jr., D.E. Némethy G. Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits.Biochemistry. 1966; 5: 365-385Crossref PubMed Google Scholar), remains a postulation. One key validation of the MWC theorem—measuring directly from the intermediate activation states—has been lacking, because of cooperativity among subunits that makes transitional states intrinsically unstable. Here, we designed methods locking TRPV1 in each of the intermediate activation states with varying ligand occupations, which allowed direct measurements of their thermodynamic properties under physiological conditions at equilibrium. Near-perfect equivalence in subunit contribution to allosteric coupling was confirmed. All RTX-induced open states are found to produce identical single-channel current amplitudes, suggesting that the activated pore adopts near identical conformations. Therefore, pore opening by RTX appears to be driven by a concerted conformational change. Our functional data hence suggest that structural asymmetry of the activated states occurs mainly in the vanilloid-binding pockets, consistent with the recently reported RTX–TRPV1 complex structures (16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Based on the 1.86 kcal/mol/subunit activation energy, channels with one to four bound RTX molecules are expected to exhibit a Po of 13.9%, 78.9%, 98.8%, and 99.9%, respectively. Channels with two bound RTX molecules already spend two-third of the time in the open state under physiological conditions. Only the fully RTX-bound channels were assigned an open state in cryo-EM studies (16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 28Gao Y. Cao E. Julius D. Cheng Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.Nature. 2016; 534: 347-351Crossref PubMed Google Scholar), suggesting a shift in stability of the closed and/or open conformation under these experimental conditions, such as lower temperatures (22Yang F. et al.The conformational wave in capsaicin activation of transient receptor potential vanilloid 1 ion channel.Nat. Commun. 2018; 9: 1-9PubMed Google Scholar). Concerted pore opening has been previously proposed for other channel types including Kv channels, for which independent voltage-sensor movements in the four subunits precede channel activation (29Hoshi T. Zagotta W.N. Aldrich R.W. Biophysical and molecular mechanisms of shaker potassium channel inactivation.Science. 1990; 250: 533-538Crossref PubMed Google Scholar, 30Schoppa N.E. Sigworth F.J. Activation of shaker potassium channels. II. Kinetics of the V2 mutant channel.J. Gen. Physiol. 1998; 111: 295-311Crossref PubMed Scopus (68) Google Scholar). Very brief (microseconds) subconductance states were found to be associated with asymmetrical subunit activations by voltage, hence representing intermediate gating states (31Zheng J. Vankataramanan L. Sigworth F.J. Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.J. Gen. Physiol. 2001; 118: 547-564Crossref PubMed Scopus (34) Google Scholar, 32Zheng J. Sigworth F.J. Selectivity changes during activation of mutant shaker potassium channels.J. Gen. Physiol. 1997; 110: 101-117Crossref PubMed Scopus (117) Google Scholar). It was proposed that subconductance states could arise from filtering effects on very rapid flickering events—representing likely a concerted transition—between the fully open state and the closed state, with voltage-dependent subunit activations progressively shifting the equilibrium toward the open state (31Zheng J. Vankataramanan L. Sigworth F.J. Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.J. Gen. Physiol. 2001; 118: 547-564Crossref PubMed Scopus (34) Google Scholar). While TRPV1 activation by RTX exhibited similar progressive shifting toward the open state, dwell times in the closed and open states appeared to be long enough to skip the filtering effect on current amplitude. The allosteric behavior described in the present study may help correlate published high-resolution channel structures (16Zhang K. Julius D. Cheng Y. Structural snapshots of TRPV1 reveal mechanism of polymodal functionality.Cell. 2021; 184: 5138-5150.e12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 33Kwon D.H. Zhang F. Fedor J.G. Suo Y. Lee S.-Y. Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis.Nat. Commun. 2022; 13: 1-12Crossref PubMed Scopus (4) Google Scholar) with distinct functional states toward a better understanding of the activation mechanism of TRPV1 and other ion channels. The mouse TRPV1 wildtype and Y512A mutant complementary DNAs (cDNAs) were constructed into pEYFP-N3 vector, where the C terminus of the channel was fused with the cDNA encoding enhanced YFP to help identify transfected cells during patch-clamp experiments, as previously described (34Cheng W. Yang F. Takanishi C.L. Zheng J. Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties.J. Gen. Physiol. 2007; 129: 191-207Crossref PubMed Scopus (154) Google Scholar). The Y512A point mutation was introduced using a mutagenesis kit (Agilent Technologies) and confirmed by sequencing. Concatemers YYYY, YYYA, YYAA, YAYA, AYAY, YAAA, and AAAA, representing tandem tetrameric cDNA constructs of rat TRPV1 wildtype (Y) and Y511A (A, equivalent to Y512A in mouse channel), were generous gifts from Dr Avi Priel and have been previously described (21Hazan A. Kumar R. Matzner H. Priel A. The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.Sci. Rep. 2015; 5: 1-13Crossref Scopus (37) Google Scholar). Human embryonic kidney 293T cells (purchased from American Type Culture Collection) were cultured in a Dulbecco's modified Eagle's medium (Hyclone or Gibco) supplemented with 10% (v/v) fetal bovine serum (Corning or GenClone), 1% (v/v) penicillin/streptomycin (Fisher Scientific), and 1% (v/v) MEM Nonessential Amino Acids Solution (Hyclone) at 37 °C with 5% CO2. Cells were cultured onto 25 mm glass coverslips (Fisher Scientific) in 35 mm chambers 18 to 24 h before transfection. Transient transfection was conducted 18 to 24 h before patch-clamp recording using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For macroscopic recordings, 1 μg plasmid was used for transfection for each chamber; for single-channel recordings, 0.1 μg plasmid was used. For concatemer channels without a fluorescent tag, the YFP plasmid was cotransfected (0.2 μg) for each chamber. Patch-clamp recordings were done using an EPC10 amplifier controlled with PatchMaster software (HEKA) in configurations as specified in the Results section. All single-channel recordings were from inside–out patches; most of the macroscopic recordings were also from inside–out patches; whole-cell recordings were collected to increase the macroscopic current level, especially for mutants such as E571A mTRPV1, E571AT551A mTRPV1, E571AI574A mTRPV1, because of their poor expression levels. Patch pipettes were pulled from borosilicate glass (A-M systems or Sutter Instrument) using Sutter Instrument P-97 micropipette puller and fire-polished to 2 to 6 MΩ for macroscopic recordings or 8 to 15 MΩ for single-channel recordings. For whole-cell recordings, serial resistance was compensated by 60%. Current signal was filtered at 2.3 to 2.9 kHz and sampled at 10.0 to 12.5 kHz. All recordings were conducted at room temperature. Ruthenium red (10 μM) and/or Ba2+ (100 mM) was used to block channel current for checking leak. A holding potential of 0 mV was used, from which a 300 ms step to +80 mV followed by a 300 ms step to −80 mV was applied. The durations of the +80 mV and −80 mV steps were between 300 ms and 2 s, adjusted according to experimental needs. The voltage was held at +80 mV for long continuous single-channel recordings. Standard symmetrical bath and pipette solutions contained (millimolar) 140 NaCl, 0.2 EGTA, 10 glucose (optional), and 15 Hepes (pH 7.2 to 7.4). Solution switching was achieved with a Rapid Solution Changer (RSC-200; Biological Science Instruments). 2-APB was dissolved in dimethyl sulfoxide to make 1 M stock solution and diluted to working concentrations using the bath solution; CAP was dissolved in dimethyl sulfoxide to make 50 mM stock solution and diluted to working concentrations using the bath solution; RTX was dissolved in ethanol to make 1 mM stock solution and diluted to working concentrations using the bath solution. To obtain single-channel recordings with a fixed number of RTX molecules in the concatemers (YYYY, YYYA, YYAA, YAYA, AYAY, YAAA, and AAAA), 3 mM 2-APB was first perfused onto an inside–out patch to confirm the number of channels in the patch. A 100 nM RTX solution was then perfused onto the patch until Po reached the stable maximal level to make sure all the binding sites were saturated by RTX. The bath solution was perfused to wash off the reversibly bound RTX until Po reached a stable level. This Po level was regarded as the Po value when the channel was bound with 0 to 4 RTX molecules according to different concatemers (4 for YYYY; 3 for YYYA; 2 for YYAA, YAYA, and AYAY; 1 for YAAA; and 0 for AAAA). To evaluate RTX binding to TRPV1, we used the cryo-EM structures of TRPV1 solved in complex with RTX in the open (O1 state, Protein Data Bank [PDB]: 7L2L) and closed (C1 state, PDB: 7L2N; C2 state, PDB: 7MZ5) states to model RTX interaction. RosettaLigand (RosettaCommons) (19DeLuca S. Khar K. Meiler J. Fully flexible docking of medium sized ligand libraries with rosettaligand.PLoS One. 2015; 10e0132508Crossref Scopus (69) Google Scholar) was used to dock RTX to wildtype and Y511A mutant structures for each state. We generated 2000 RTX conformers for the docking process using BioChemical Library (20Brown B.P. et al.Introduction to the BioChemical Library (BCL): an application-Based open-source toolkit for integrated cheminformatics and machine learning in computer-aided drug discovery.Front. Pharmacol. 2022; 13833099Crossref Scopus (3) Google Scholar). The detail of the docking algorithm has been described elsewhere (19DeLuca S. Khar K. Meiler J. Fully flexible docking of medium sized ligand libraries with rosettaligand.PLoS One. 2015; 10e0132508Crossref Scopus (69) Google Scholar) (see Appendix for Rosetta docking scripts and command lines). A total of 10,000 docking models were generated, and the top 1000 models ranked by total_score were selected for analysis. Binding energy is represented by the interface_delta_X term reported in Rosetta energy unit. All-atom RMSD was used to compare the generated docking RTX models to the RTX-binding conformations captured in the cryo-EM structures. Patch-clamp data were exported and analyzed using Igor Pro 8 (WaveMetrics). Statistical analyses were done using GraphPad Prism 8 (GraphPad Software, Inc). Macroscopic current amplitude was calculated by measuring the difference between ligand-activated current and the baseline current level before any ligand perfusion. A digital filter at 0.4 kHz was used for analyzing single-channel amplitude and Po. Single-channel recordings were analyzed using all-point histograms and fitted to a double-Gaussian function; single-channel amplitude was measured as the difference between the two gaussian peaks; single-channel Po was measured by calculating the portion of open events over the total time recorded. Only true single-channel recordings (n = 22) or two-channel recordings (n = 2) were used. Po of two-channel recordings was calculated using the equation Po=t1+2t22T, where t1 is the total time of one-channel opening events observed, t2 is the total time of two-channel opening events observed, T is the total recording time. Spontaneous Po was measured using the equation Po=tNT, where t is the total time of the open events observed at room temperature in bath solution, N is the total number of channels in the patch, which was determined by calculating the amplitude ratio between maximum current and the average single-channel current, T is the total recording time. There were no overlapping open events in these recordings. To analyze single-channel dwell times, the recorded single-channel traces were processed using the single-channel search function in Clampfit (Molecular Devices) to generate dwell-time histograms (35Sigworth F. Sine S. Data transformations for improved display and fitting of single-channel dwell time histograms.Biophys. J. 1987; 52: 1047-1054Abstract Full Text PDF PubMed Google Scholar). Model fitting was conducted in Igor, using the global fitting procedure when needed. Statistical analysis was done using GraphPad Prism 8. Student’s t test was used when comparing between two groups. For comparisons within the same recordings, paired t test was used. For comparison between multiple groups, one-way ANOVA with Tukey’s multiple comparisons test was used. All data generated or analyzed during this study are included in the article and supporting information. This article contains supporting information. The authors declare that they have no conflicts of interest with the contents of this article. We are grateful to Avi Priel for sharing the concatemer constructs. We thank members of the Zheng and Yarov-Yarovoy laboratories for assistance and discussion. S. L. and J. Z. conceptualization; S. L. and P. T. N. methodology; S. L. and J. Z. writing–original draft; S. L., P. T. N., S. V., V. Y.-Y., and J. Z. writing–review & editing; J. Z. and V. Y.-Y. supervision; J. Z. and V. Y.-Y. funding acquisition. National Institutes of Health grant R01NS103954 (to J. Z. and V. Y.-Y.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) are commonly found in the environment as components of brominated flame retardants. Due to their potential impact on human health and wildlife, it is imperative to closely monitor and manage their levels in the environment. This study investigated the spatial distribution, sources, and ecological risks of PBDEs and HBCDs in Jiaozhou Bay (JZB), a large bay situated on the eastern coast of China. The results showed that PBDE concentrations ranged from not detected (ND) to 7.93 ng/L in the water and ND to 65.76 ng/g in the sediment, while HBCD concentrations ranged from ND to 0.31 ng/L in the water and ND to 16.63 ng/g in the sediment. Furthermore, we observed significantly higher concentrations of PBDEs and HBCDs in the inner JZB compared to the outer JZB. Our source apportionment analysis showed that PBDEs primarily originated from the production and debromination of BDE-209, as well as the emission of commercial PeBDEs, whereas HBCDs in sediments mostly stemmed from anthropogenic activities and river input. Finally, our eco-logical risk assessment highlighted the need for continuous monitoring of PBDEs in JZB sediments. Overall, our study aims to provide valuable assistance for the environmental management of the JZB bay area, which is characterized by a complex net-work of rivers and a thriving economy.

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that is regulated by multiple facilitators, such as Rad26, and repressors, such as Rpb4 and Spt4/Spt5. How these factors interplay with each other and with core RNA polymerase II (RNAPII) remains largely unknown. In this study, we identified Rpb7, an essential RNAPII subunit, as another TCR repressor and characterized its repression of TCR in the AGP2, RPB2, and YEF3 genes, which are transcribed at low, moderate, and high rates, respectively. The Rpb7 region that interacts with the KOW3 domain of Spt5 represses TCR largely through the same common mechanism as Spt4/Spt5, as mutations in this region mildly enhance the derepression of TCR by spt4Δ only in the YEF3 gene but not in the AGP2 or RPB2 gene. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII repress TCR largely independently of Spt4/Spt5, as mutations in these regions synergistically enhance the derepression of TCR by spt4Δ in all the genes analyzed. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII may also play positive roles in other (non-NER) DNA damage repair and/or tolerance mechanisms, as mutations in these regions can cause UV sensitivity that cannot be attributed to derepression of TCR. Our study reveals a novel function of Rpb7 in TCR regulation and suggests that this RNAPII subunit may have broader roles in DNA damage response beyond its known function in transcription.

Nucleosome structure and repair ofN-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhancedN-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally,N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells. Nucleosome structure and repair ofN-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhancedN-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally,N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells. Simple methylating agents, such as methyl methanesulfonate (MMS) 1The abbreviations used are: MMS, methyl methanesulfonate; BER, base excision repair; BLM, bleomycin; DMS, dimethyl sulfate; 3MeA, N 3-methyladenine; 7MeG, N 7-methylguanine; NER, nucleotide excision repair; NMP, N-methylpurine; TS, transcribed strand; NTS, nontranscribed strand; UAS, upstream-activating sequence1The abbreviations used are: MMS, methyl methanesulfonate; BER, base excision repair; BLM, bleomycin; DMS, dimethyl sulfate; 3MeA, N 3-methyladenine; 7MeG, N 7-methylguanine; NER, nucleotide excision repair; NMP, N-methylpurine; TS, transcribed strand; NTS, nontranscribed strand; UAS, upstream-activating sequence and dimethyl sulfate (DMS), produce a variety of damaged bases in DNA of whichN7-methylguanine (7MeG) andN3-methyladenine (3MeA) constitute ∼80 and 10%, respectively (1Singer B. Grunberger D. Molecular Biology of Mutagens and Carcinogens. Plenum Press, NY1983: 55-78Google Scholar, 2Pieper R.O. Nickoloff J.A. Hoekstra M.F. DNA Damage and Repair, Vol. 2: DNA Repair in higher eukaryotes. Humana Press Inc., Totowa, NJ1998: 199-222Google Scholar). These N-methylpurines (NMPs), can be enzymatically removed or they can spontaneously depurinate to produce abasic sites, which may be more mutagenic than NMPs (3Posnick L.M. Samson L.D. Mutat. Res. 1999; 257: 127-143Google Scholar, 4Xiao W. Chow B.L. Hanna M. Doetsch P.W. Mutat. Res. 2001; 487: 137-147Google Scholar). Some DNA lesions that are repaired by nucleotide excision repair (NER) or certain base excision repair (BER) pathways are removed much faster in the transcribed strand (TS) than in the nontranscribed strand (NTS) of an active gene (5Hanawalt P.C. Mutat. Res. 2001; 485: 3-13Google Scholar). In contrast, repair of NMPs does not appear to be coupled to transcription, since the TS and NTS have similar repair rates in the genes analyzed to date (6Scicchitano D.A. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3050-3054Google Scholar, 7Ye N. Holmquist G.P. O'Connor T.R. J. Mol. Biol. 1998; 284: 269-285Google Scholar, 8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). However, repair rates of NMPs vary dramatically at different sites in both the humanPGK1 gene (7Ye N. Holmquist G.P. O'Connor T.R. J. Mol. Biol. 1998; 284: 269-285Google Scholar) and the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). One reason for this repair heterogeneity is the effect of nearest-neighbor nucleotides (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar), in that slow repair occurs at NMPs between purines and fast repair occurs at NMPs between pyrimidines. It was proposed that different stabilities of base stacking between adjacent base pairs can affect flipping out of NMPs from the DNA helix during recognition and incision by DNA glycosylase (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). In the nucleus of eukaryotic cells, DNA is packaged into a nucleoprotein complex known as chromatin (9Wolffe A.P. Chromatin: Structure and Function. 3rd Ed. Academic Press, London, and New York1999: 7-172Google Scholar). This complex provides the compaction and structural organization of DNA for processes such as replication, transcription, recombination, and repair. The fundamental subunits of chromatin are nucleosome cores, where 147 bp of DNA is wrapped around a histone octamer (10Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Google Scholar). DNA between two adjacent nucleosome cores is called linker DNA, which varies in length from about 20 to 90 bp in different organisms and tissues, or between individual nucleosomes in the same cell (11van Holde K.E. Chromatin. Springer-Verlag KG, Berlin1989: 289-354Google Scholar). The effect of nucleosome structure on NMP repair is not understood. In rat liver cells, it was shown that the overall removal of NMPs occurred at a relatively uniform rate in different chromatin fractions (i.e. active chromatin, bulk genome and nuclear matrix) (12Ryan A.J. Billett M.A. O'Connor P.J. Carcinogenesis. 1986; 7: 1497-1503Google Scholar). On the other hand, within the yeast minichromosome YRpSO1, there was a mild correlation between repair rates and nucleosome positioning in regions of the inducible GAL1:URA3 fusion gene, but not in the constitutively expressed HIS3 gene on the same plasmid (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). To further address questions on the effect of nucleosome structure on repair of NMPs in intact cells, we examined repair in the divergent yeast GAL1-10 genes, which share a common upstream-activating sequence (UAS). These genes are induced to very high levels of expression in galactose, but are completely repressed in glucose (13Bash R. Lohr D. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 197-259Google Scholar). Extensive studies have been done on isolated nuclei or chromatin to map the nucleosome structure in the GAL1-10region and the effects of galactose induction (14Lohr D. Torchia T. Hopper J. J. Biol. Chem. 1987; 262: 15589-15597Google Scholar, 15Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar, 16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar, 17Cavalli G. Thoma F. EMBO J. 1993; 12: 4603-4613Google Scholar). However, it is possible that subtle changes in nucleosome structure of this region differ in whole cells, and were missed because of the procedure of nuclei and/or chromatin isolation. Thus, to examine the influence of nucleosome structure on repair of NMPs in intact cells, we developed a nucleosome mapping procedure using bleomycin (BLM), which avoids isolation of nuclei or chromatin. This basic glycopeptide-derived antibiotic has been shown to preferentially cleave nucleosome linker DNA in isolated Chinese hamster nuclei (18Kuo M.T. Hsu T.C. Nature. 1978; 271: 83-84Google Scholar), lysophosphatidylcholine-permeabilized human cells (19Sidik K. Smerdon M.J. Cancer Res. 1990; 50: 1613-1619Google Scholar), and whole yeast cells (20Moore C.W. Cancer Res. 1988; 48: 6837-6843Google Scholar). However, BLM has not been used to map nucleosome positions in specific sequences in whole cells, since highly specific cleavage in linker DNA has not been achieved. By using the mild nonionic detergent digitonin to efficiently permeabilize yeast cells, and rich medium to effectively stop BLM cleavage during DNA isolation, we were able to map nucleosome structure at nucleotide resolution in the GAL1-10region in whole yeast cells and directly correlate repair of NMPs with nucleosome structure. DBY747 (MATa ura3-52his3-Δ1 leu2–3 leu2–112 trp1–289) and its isogenic mutant strains JC8901 (mag1Δ::hisG- URA3-hisG), WXY9379 (rad1Δ::LEU2), and WXY9380 (mag1Δ::hisG- URA3-hisG rad1Δ::LEU2) were generously provided by Dr. Wei Xiao (University of Saskatchewan, Canada). Strain Y452 (MATα ura3-52 his3–1 leu2–3 leu2–112) was provided by Dr. Louise Prakash (University of Texas Medical Branch, Galveston, TX). Yeast cells were grown at 30 °C in minimal medium containing 2% glucose or 2% galactose to late-log phase (OD600 ∼1.0). After washing twice with ice-cold 2% glucose (for glucose cultures) or 2% galactose (for galactose cultures), the cells were resuspended in 50 mm NaCl, 2 mm MgCl2, 0.02% glucose or galactose, to give a cell density of 2 × 109cells/ml. Digitonin (Sigma, 10% stock) and Fe(NH4)2(SO4)2 (10 mm, freshly dissolved in H2O) were mixed with the cell suspension to give a final concentration of 0.05% and 50 μm, respectively. BLM (Sigma, 20 units/ml stock) was then added to final concentrations of 0–400 milliunits/ml, and the mixture was incubated at 30 °C for 12 min. To stop the reaction, the cell suspension was mixed with 100 volumes of ice-cold YPD (1% yeast extract, 2% peptone, 2% glucose) or YPG (1% yeast extract, 2% peptone, 2% galactose) and pelleted by centrifugation. For the second wash, cells were resuspended in ice-cold 2% glucose or 2% galactose, mixed with 1:10 volume of a stock solution containing 10% yeast extract and 20% peptone, and pelleted by centrifugation. It has been shown that BLM associates with outer plasma membranes of mammalian cells (CV-1), and can cleave DNA after the cells are lysed, even in the presence of EDTA (21Solomon L.R. Beerelli R.D. Moseley P.L. Biochemistry. 1989; 28: 9932-9937Google Scholar). Furthermore, BLM molecules can only be efficiently removed from mammalian cell membranes by trypsin treatment (21Solomon L.R. Beerelli R.D. Moseley P.L. Biochemistry. 1989; 28: 9932-9937Google Scholar). We found BLM can be efficiently removed after treatment, simply by washing the cells with media containing yeast extract and peptone. The naked DNA used in these experiments was a PCR fragment of the yeastGAL1-10 region. Reactions (50 μl) contained 50 mm Tris-HCl (pH 8.0), 100 ng of a PCR product, 1 μg of sonicated salmon sperm DNA, 1–5 μm of freshly prepared Fe(NH4)2(SO4)2 and 0–5 milliunits/ml of BLM. After 12 min of incubation at 30 °C, the DNA was quickly separated from the reaction mixture with the QIAquick Nucleotide Removal Kit (Qiagen). Yeast cells were grown at 30 °C in minimal medium containing 2% glucose or 2% galactose to late log phase (OD600 ∼1.0), and mixed with DMS (Sigma, undiluted solution) to give a final concentration of 0.03% (v/v). After 2 min incubation at room temperature, cells were washed twice with ice-cold 2% glucose (or 2% galactose) and resuspended in the same solutions containing 100 mmhydroxyurea, to prevent DNA replication during repair incubation (22Slater M.L. J. Bacteriol. 1972; 113: 263-270Google Scholar). One-tenth volume of a solution containing 10% yeast extract and 20% peptone was added to the DMS-treated cultures. After different times of repair incubation at 30 °C, an aliquot was removed and put on ice. After BLM treatment or repair incubation following DMS treatment, pellets of 2 × 109 cells, were mixed with 2 ml of ice-cold nuclei isolation buffer (NIB: 50 mm Tris-HCl, 2 mmMgCl2, 150 mm NaCl, 17% glycerol, 0.5 mm spermine, 0.15 mm spermidine, pH8.0) and 2 ml of acid-washed glass beads (Sigma, 425–600 microns). The mixtures were vortexed for 30 s and kept on ice for 2–5 min. This procedure was repeated three times to completely break up the cells. The samples were mixed with 10 ml of 50 mm Tris-HCl, 400 mm NaCl, 2% SDS, 2 mm EDTA, pH 8.0, and incubated at 65 °C for over 30 min. After cooling to room temperature, the samples were mixed with 8 ml of 5 m NaCl and left on ice overnight. The samples were then centrifuged at 4 °C, and the supernatant was collected. The DNA was precipitated, treated with RNase A, and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). After re-precipitation, the DNA was dissolved in H2O and stored at −20 °C before using. The sites of BLM cleavage and NMPs were mapped along a 1.9-kb region encompassing the common UAS and 5′-portion of the GAL1 and GAL10genes. Five overlapping restriction fragments (four of these fragments are shown in Figs. 1 and 2) on each strand of the region were end-labeled using the procedure described previously (23Li S. Waters R. Carcinogenesis. 1996; 17: 1549-1552Google Scholar, 24Li S. Waters R. Smerdon M.J. Methods: Companion Methods Enzymol. 2000; 22: 170-179Google Scholar), with slight modification. Briefly, about 2–3 μg of genomic DNA was digested with restriction endonuclease(s) to release the fragments of interest. For NMP mapping, the restricted DNA was further cleaved at the NMP sites by incubating DNA in 1 m piperidine at 90 °C for 30 min, and the piperidine removed by evaporation (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). Excess copies of a biotinylated oligonucleotide, which has a portion complementary to one end of the fragment to be labeled, were mixed with the sample. The T tract in the biotinylated oligonucleotides described formerly (23Li S. Waters R. Carcinogenesis. 1996; 17: 1549-1552Google Scholar, 24Li S. Waters R. Smerdon M.J. Methods: Companion Methods Enzymol. 2000; 22: 170-179Google Scholar) was changed to short runs (4Xiao W. Chow B.L. Hanna M. Doetsch P.W. Mutat. Res. 2001; 487: 137-147Google Scholar, 5Hanawalt P.C. Mutat. Res. 2001; 485: 3-13Google Scholar) of Ts separated by Gs. This change ensured full-length incorporation of radioactive dAMPs opposite the Ts, and eliminated hybridization between the oligonucleotides and contaminating poly(A) tails of mRNA. The mixture was heated to 95 °C for 5 min to denature the DNA and then cooled to an annealing temperature. The annealed fragments were attached to streptavidin magnetic beads (Dynal), and the other fragments were removed by washing the beads at the annealing temperature. The attached fragments were labeled using [α-32P]dATP (PerkinElmer Life Sciences) and non-radioactive dCTP, rather than [α-32P]dATP alone (23Li S. Waters R. Carcinogenesis. 1996; 17: 1549-1552Google Scholar, 24Li S. Waters R. Smerdon M.J. Methods: Companion Methods Enzymol. 2000; 22: 170-179Google Scholar). The labeled fragments were resolved on sequencing gels and exposed to PhosphorImager screens (Molecular Dynamics).Figure 2Bleomycin cleavage of the bottom strand of the GAL1-10 region ( NTS for GAL10 and TS for GAL1). Panels are of sequencing gels for four of five overlapping restriction fragments analyzed along a 1.9-kb region of the GAL1-10 genes. See legend to Fig. 1for details.View Large Image Figure ViewerDownload (PPT) Sequence ladders were generated from PCR products of theGAL1-10 fragments, using the rapid Maxam-Gilbert method (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.1989: 13.95-13.97Google Scholar). The ladders were labeled using the same procedure as that for the BLM or DMS treated DNA samples. The BLM cleavage at individual sites is reflected by the band intensity on a gel. The doses of BLM used were relatively low, to ensure that cleavage by BLM followed single-hit kinetics for the majority of fragments analyzed. However, even under these conditions, a small portion of the fragments still have more than one cleavage, and a small portion of the signal at a site will not show up on a gel if the cleavage occurs upstream (relative to the labeled end) of the site. The effect of this “hidden signal” increases as a cleave site is more distant from the labeled 3′-end, which runs at the bottom of a gel. For a fragment of ≈400 nucleotides (i.e. the maximum length quantified on the sequencing gels), the hidden signal at a site close to the top of a gel can be as much as 30% of the total signal in a band. To correct for this, the following algorithm was used: Let A denote the total signal intensity in a gel lane, X the actual signal intensity (i.e. not reduced by upstream cleavages) at position N in a gel lane, and C the total signal intensity upstream of position N (the upstream sites run below position N in a gel lane). The proportion of signal intensity at position N is X/A and the proportion of signal intensity upstream of position N is C/A. Suppose BLM cleavage at specific sites is randomly distributed among different fragments (i.e. follow a Poisson distribution), then the probability of cleavages at position N and those upstream of position N on the same fragments is (X/A) × (C/A). Therefore, the total number of cleavages at position N and those upstream of position N on the same fragment is A × [(X/A) × (C/A)], or (X × C)/A. If B denotes the observed signal intensity at position N on the gel, then (X × C)/A = X − B and the actual signal intensity (X) at position N is (A × B)/(A − C). The signal intensity at all pixels in a gel lane was measured by ImageQuaNT software (Molecular Dynamics) and the data transferred to Microsoft Excel. After the gel background signal was subtracted, the total signal intensities in different lanes of a gel were normalized to the same amount. The signal intensity at all pixels along a gel lane (including the control lanes not cleaved with BLM) was then corrected using the above described equation X = (A × B)/(A − C). The signal intensities in a lane containing the uncleaved sample were used as a baseline for other lanes that contain BLM cleaved samples. As the signal intensities along a gel lane of uncleaved sample fluctuate, the baseline was smoothed by local averaging of signal intensities (at continuous intervals of 100–200 pixels) in the lane. The corrected signal intensities in a lane containing BLM cleaved sample were then corrected by subtraction of the smoothed baseline. The data was then imported to PeakFit (SPSS, Inc.) to fit and deconvolute peaks corresponding to the individual bands on the gels (24Li S. Waters R. Smerdon M.J. Methods: Companion Methods Enzymol. 2000; 22: 170-179Google Scholar). Quantitation for NMPs followed the same procedure as that for BLM cleavage. Wild type (Y452) yeast cells were grown in glucose or galactose medium, permeabilized with digitonin and treated with BLM. The time used for digitonin permeabilization and BLM treatment was 12 min, which is just long enough to see noticeable BLM cleavage in chromatin DNA. Under these conditions, different concentrations of BLM showed a linear response of band intensity (Figs. 1 and2 and data not shown). Therefore, the cleavage by BLM followed single-hit kinetics for the majority of the chromatin DNA fragments. Genomic DNA was isolated from the BLM-treated cells and cut with restriction enzyme to release the fragments of interest. A total of 5 overlapping restriction fragments were analyzed for each strand of theGAL1-10 region. The restricted fragments were strand-specifically end-labeled, resolved on DNA sequencing gels, and exposed to phosphorimager screens. For comparison, naked DNA of the same GAL1-10 region was also treated in parallel with the chromatin DNA. As can be seen from the gels in Figs. 1 and 2, naked DNA was cleaved at very low concentrations of BLM (5 milliunits/ml) and Fe2+ (1 μm), and no cleavage was observed in the presence of BLM or Fe2+ alone (lanes 1 and2 in Figs. 1 and 2; data not shown). Almost all pyrimidines 3′ to guanines are cleaved by BLM (compare lanes 2 in each gel with the sequencing lanes C+T and G) consistent with previous reports (26D'Andrea A.D. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3608-3612Google Scholar). Most adenines 3′ to guanines are also cleaved, but generally to a lesser extent (Figs. 1 and 2,arrowheads to the right of gels). Furthermore, some sites of weak cleavage are also observed at pyrimidines 3′ to adenines (Figs. 1 and 2, small horizontal bars on theright side of gels). Much higher concentrations of BLM and Fe2+ are needed for cleavage of chromatin DNA in permeabilized cells to reach the same level as that for naked DNA (Figs. 1 and 2). Furthermore, there was consistently more cleavage of chromatin DNA in glucose cultures than in galactose cultures, when the same concentration of BLM was applied (Figs. 1 and 2; data not shown). Most likely, BLM is hydrolyzed more rapidly in galactose cultures due to the induction of BLM hydrolase, which is encoded by the GAL6 gene (27Zheng W., Xu, H.E. Johnston S.A. J. Biol. Chem. 1997; 272: 30350-30355Google Scholar). To determine the protection level of a nucleosome to its DNA, the ratios of band intensities for BLM cleavage of naked DNA to chromatin DNA (in glucose and galactose cultures) were determined from scans of phosphorimages and peak deconvolution (24Li S. Waters R. Smerdon M.J. Methods: Companion Methods Enzymol. 2000; 22: 170-179Google Scholar). This ratio was designated as 1.0 at a BLM cleavage site (nucleotide 1003 relative to the uniqueStuI site in the GAL10 gene, Fig. 2 C) on the bottom strand near the UAS. The ratios at other cleavage sites were then normalized to the ratio at this site. The normalized ratios are plotted in Fig. 3 A for both glucose (open triangles) and galactose (solid triangles) grown cells, and the smoothed curves (by averaging values in continuous intervals of 40 nucleotides) for these values are plotted in Fig. 3 B. The BLM cleavage pattern at most sites in the UAS region is similar between chromatin (both glucose and galactose cultures) and naked DNA (Figs. 1 C and 2 C), suggesting the UAS region is nucleosome-free in both glucose and galactose cultures. This is in agreement with DNase I (28Lohr D. Nucleic Acids Res. 1984; 12: 8457-8474Google Scholar) and (methiumpropyl−EDTA)iron(II) (16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar) mapping in isolated nuclei. However, a few sites in the UAS region do show inhibition or enhancement of BLM cleavage (Figs. 1 C and 2 C, UAS region), indicating the binding of nonhistone protein(s), most notably Gal4, to this region may modulate BLM cleavage. In glucose cultures, where the genes are repressed, a region of about 150 bp on each side of the UAS is strongly protected from BLM cleavage (Figs. 1, B and C; 2, B andC; and 3, see regions marked by ovals e andf), indicating that each of these regions contains a nucleosome with a fixed position on DNA. The protection of flanking ∼150 bp regions (Figs. 1-3, ovals d and g) is less apparent, and the protection diminishes with increasing distance from the UAS (qualitatively represented by the shading of ovals in Figs. 1-3). Indeed, the protection levels can be 10-fold greater in the core regions of nucleosomes e and f (Figs.1-3), which are adjacent to the UAS, than those of nucleosomesa, b, c, h, andi, which are farther away from the UAS. These results indicate that the variability of nucleosome positions sharply increases with increasing distance from the UAS in intact cells. In galactose cultures, where GAL1-10 genes are induced, all the nucleosome core sequences in the GAL1-10 region analyzed are less protected from BLM cleavage (Figs. 1-3), indicating nucleosomes are disrupted upon galactose induction. This disruption is most striking in nucleosomes e and f, which are adjacent to the UAS, presumably due to their occupying the minimum number of positions in glucose cultures (Figs. 1-3). The same wild type (Y452) yeast cells as those used for nucleosome mapping were used for analyzing NMP induction and repair in the GAL1-10 region. The cells were treated with 0.03% DMS for 2 min to induce NMPs (see “Experimental Procedures”). After different times of repair incubation, genomic DNA was isolated, digested with restriction enzymes, and cleaved at NMPs by hot alkaline treatment. The cleaved fragments were strand-specifically end-labeled, resolved on DNA sequencing gels and exposed to phosphorimager screens. As can be seen from the gels in Figs. 4 and5, the main type of NMPs induced is 7MeG (e.g. compare 0-h lanes with G lanes), while 3MeA is induced to a much lower extent (e.g. compare0 h lanes with G and GA lanes). This pattern of NMP induction is similar to that seen with the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar) and to that of other past reports (2Pieper R.O. Nickoloff J.A. Hoekstra M.F. DNA Damage and Repair, Vol. 2: DNA Repair in higher eukaryotes. Humana Press Inc., Totowa, NJ1998: 199-222Google Scholar).Figure 5Induction and repair of NMPs in the bottom strand ( NTS for GAL10 and TS forGAL1) of the GAL1-10 region for glucose (Glu) and galactose (Gal) cultures. See the legend to Fig.4 for details.View Large Image Figure ViewerDownload (PPT) In galactose cultures, a strong protection from DMS methylation is seen at Gs in the triplet sequences of CGG, AGG, or CGC located at both ends of the palindromic Gal4 binding sites (brackets in Figs. 4 Cand 5 C; Gs marked with arrowheads on theright side of gels), consistent with a previous report (29Giniger E. Varnum S.M. Ptashne M. Cell. 1985; 40: 767-774Google Scholar). Meanwhile, an enhancement of methylation can also be seen in some sites of the UAS region, especially in the top strand (Figs. 4 Cand 5 C, small horizontal bars on the right side of gels). Moreover, there is a difference between glucose and galactose cultures in NMP yield at several sites in the promoter regions of the two genes (Figs. 4 B and 5 B, stars on the right side of gels). On the other hand, NMP yields at most sites throughout the region are similar between the two cultures (Figs. 4 and5), indicating that the presence of nucleosomes does not markedly affect NMP induction. This agrees with a previous in vitrostudy showing that formation of nucleosomes does not significantly modulate NMP induction by DMS (30McGhee J.D. Felsenfeld G. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2133-2137Google Scholar). The repair rates of NMPs at different sites were dramatically different (>30-fold at sites) (Figs. 4-6). As can be seen from the gels in Figs. 4 and 5, most NMP sites located between pyrimidines (C or T) were repaired much faster than NMPs between purines (A or G), and those located between a purine and a pyrimidine were repaired at intermediate rates. In order to assess the generality of these observations, the percentages of 7MeGs remaining following different times of repair incubation in the same contexts of nearest-neighbor nucleotides (i.e. between purines, pyrimidines, and between a purine and a pyrimidine) were averaged. As can be seen from Fig. 7, the statistical data confirm these observations. As limited 3MeA sites were available for analysis, the effect of the nearest-neighbor nucleotides on 3MeA repair cannot be analyzed by this method.Figure 7Effect of nearest-neighbor nucleotides on repair of 7MeGs in the GAL1-10 genes of glucose (A) and galactose (B) cultures, respectively. The data in each panel represent the mean (± 1 S.D.) of the percent of 7MeGs remaining at all the sites that could be analyzed in the 1.9-kb region between purines (—⃝—, 122 sites), pyrimidines (—△—, 137 sites), and a purine and a pyrimidine (dashed line, 259 sites). The S.D. for the 7MeG sites between a purine and a pyrimidine are not included to more clearly show the difference of repair rates between the other two contexts of nearest-neighbor nucleotides.View Large Image Figure ViewerDownload (PPT) In glucose cultures, the strongest correlation between nucleosome positioning and NMP repair can be seen in the two nucleosomes (e and f) with the most fixed positions (Figs. 4-6). In these regions, slow repair occurs in the nucleosome core DNA and faster repair takes place in nucleosome linker or nucleosome-free DNA (i.e. the UAS region). This correlation sharply fades off in the nucleosomes that are more distant from the UAS, in agreement with the observations that the variability of nucleosome positioning sharply increases with distance from the UAS. This profile of correlation can be seen more clearly after the individual times required for repairing 50% of the NMPs (T½) are smoothed (by locally averaging the values in continuous 40-nucleotide intervals) (Fig. 6 B). However, with the marked effects of nearest-neighbor nucleotides on NMP repair (see above) superimposed on the effects of nucleosome structure, the nucleosome effect is masked and more difficult to discern in the regions distant from the UAS. Galactose induction causes enhancement of repair in both strands of the nucleosome core DNA (Figs. 4-6). Indeed, increases of as much as 8-fold occur in the core regions of nucleosomes e andf, which are disrupted most dramatically (Fig.6 C). Interestingly, this galactose-enhancement of NMP repair also fades off with distance from the UAS (Fig. 6 C), correlating well with the nucleosome positioning and disruption trends (compare Figs. 3 B and 6 C). It has been shown thatmag1 mutants that are defective in NER (rad1 orrad2 mutants) are extremely sensitive to MMS-induced killing, and the effects of these mutations are synergistic (31Xiao W. Chow B.L. Curr. Genet. 1998; 33: 92-99Google Scholar). This suggests that NER may provide an alternative pathway for repair of NMPs. To assess the contribution of NER, repair of NMPs was analyzed in different genomic regions of isogenic wild type, mag1,rad1, and mag1 rad1 cells. As examples, NMP repair in regions of the GAL1 gene and the constitutively expressed RPB2 gene, which encodes the second largest subunit of RNA polymerase II, is shown in Fig.8. As can be seen, essentially normal repair occurred in rad1 cells. In contrast, deletion of theMAG1 gene almost completely abolishes repair of NMPs, although residual repair can be seen if the RAD1 gene is present in the mag1 cells (Fig. 8). Repair analysis in other genomic regions shows the same trends (data not shown), indicating NER plays little, if any, role in the repair of NMPs in S. cerevisiae. We have mapped nucleosome structure and repair ofN-methylpurines in whole yeast cells. The major nucleosome positions observed in this report agree well with past reports on isolated nuclei or chromatin (15Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar, 16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar, 17Cavalli G. Thoma F. EMBO J. 1993; 12: 4603-4613Google Scholar). However, in the present study, nucleosomes that occupy fixed positions in the GAL1-10promoter region were only observed in the regions adjacent to the UAS in glucose cultures, and the variability of positions sharply increases with increasing distance from the UAS (Figs. 1-3). This observation fits well with the finding that the binding of Y factor to a short sequence that overlaps the Gal4 binding site II of the UAS serves as a nucleosome positioning boundary (15Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar). In contrast, mapping with isolated nuclei or chromatin showed longer arrays of precisely positioned nucleosomes in the entire GAL1-10 region (15Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar, 16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar, 17Cavalli G. Thoma F. EMBO J. 1993; 12: 4603-4613Google Scholar). This difference may reflect a selection for lowest energy nucleosome positions in chromatin during nuclei isolation, compared with more dynamic features of nucleosomes that are distant from the UAS in intact cells. This notion agrees well with the NMP repair data (Figs. 4-6), as well as results of nucleotide excision repair of ultraviolet light induced cyclobutane pyrimidine dimers. 2S. Li and M. J. Smerdon, unpublished results. Our data strongly suggest that nucleosomes in intact cells inhibit NMP repair. First of all, in nucleosomes with fixed positions (especiallye and f), repair is much slower in the core DNA sequences than in linker DNA (Figs. 4-6). Secondly, induction of transcription enhanced NMP repair, with the regions close to the UAS (where nucleosome disruption is most dramatic) being most striking (Fig. 6 C). We note that, it is unlikely that this enhancement is caused by a direct coupling between transcription and repair, as no strand bias for repair is observed in these regions (Figs. 4-6). This enhancement, however, correlates well with nucleosome positioning and disruption profiles (compare Figs.3 B and 6 C). As multiple factors seem to influence repair of NMPs, nucleosome affected NMP repair may be obscured, especially in the more dynamic nucleosomes in the cell. In addition to the influence of nearest neighbor nucleotides (discussed below), the binding of nonhistone proteins may also affect NMP repair. Indeed, repair of NMPs in the UAS region was faster in glucose cultures than in galactose cultures, where the nonhistone protein Gal4 is bound to the UAS region (Figs. 4 C, 5 C, and 6). These considerations may explain why little or no correlation between NMP repair and nucleosome positioning is seen in regions distant from the UAS. In a previous report on NMP repair in the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar), we observed a mild correlation between repair rates and nucleosome positioning in some regions of the GAL1:URA3fusion gene, but not in the HIS3 gene (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). Presumably, these observations reflect differences in dynamics of nucleosome positions in these genes on the minichromosome. NER and Mag1-initiated BER are synergistic in response to MMS-induced DNA lesions (31Xiao W. Chow B.L. Curr. Genet. 1998; 33: 92-99Google Scholar), indicating a subset of these lesions may be repaired by both pathways. Furthermore, in vitro experiments with purified human 3-methyladenine-DNA glycosylase (MPG protein), which is the counterpart of yeast Mag1, show that MPG interacts with the human homologue of Rad23 (hHR23) (32Miao F. Bouziane M. Dammann R. Masutani C. Hanaoka F. Pfeifer G. O'Connor T.R. J. Biol. Chem. 2000; 275: 28433-28438Google Scholar). Importantly, this interaction elevates the rate of MPG-catalyzed excision from hypoxanthine-containing substrates (32Miao F. Bouziane M. Dammann R. Masutani C. Hanaoka F. Pfeifer G. O'Connor T.R. J. Biol. Chem. 2000; 275: 28433-28438Google Scholar). Our results with mag1 and rad1mutant cells suggest that, in S. cerevisiae, repair of NMPs is accomplished primarily by the Mag1-initiated BER pathway, and that NER contributes very little to the repair of these lesions. We also analyzed NMP repair in mutants lacking Rad7, Rad16 and Rad23 proteins, each of which is a component of the NER pathway (33Prakash S. Prakash L. Mutat. Res. 2000; 451: 13-24Google Scholar). None of these mutants showed a detectable deficiency in repair of NMPs (data not shown). Thus, it is possible that the substrate shared by NER and Mag1 initiated BER is not NMPs, but a rare DNA lesion that cannot be detected by our technique. We note, however, that a very small amount of repair of NMPs does occur in mag1 cells if Rad1 is present (Fig. 8). This residual repair may be sufficient to cause the observed synergy between NER and the Mag1 initiated BER for MMS-induced lesions. A number of organisms have a strong backup pathway for repairing NMPs. In E. coli, the AlkA and Tag proteins can initiate repair of these lesions, even though the substrate specificity differs for the two enzymes (34Thomas L. Yang C.-H. Goldthwait D.A. Biochemistry. 1982; 21: 1162-1169Google Scholar, 35Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Google Scholar). In Schizosaccharomyces pombe, NMPs may be repaired primarily through the NER pathway, rather than a BER pathway (36Memisoglu A. Samson L. J. Bacteriol. 2000; 182: 2104-2112Google Scholar). In mammalian cells, a pathway may exist that repairs 7MeG in the absence of the ordinarily used Aag DNA glycosylase (37Smith S.A. Engelward B.P. Nucleic Acids Res. 2000; 28: 3294-3300Google Scholar). However, deletion of the MAG1 gene in S. cerevisiae cells almost completely abolishes NMP repair (Fig. 8and data not shown), and no NMP repair can be seen in mag1 rad1 double deletion cells. This indicates that S. cerevisiae may lack a strong back up pathway for repairing NMPs. Finally, as observed with the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar), there was a significant correlation between the nearest neighbor nucleotides of NMP sites and the repair of NMPs in the yeast genomicGAL1-10 region. These data suggest that the same repair machinery is used for repairing NMPs in genomic and minichromosome DNA. This finding is similar to that of Sweder and Hanawalt (38Sweder K.S. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10696-10700Google Scholar), who studied repair of ultraviolet light-induced cyclobutane pyrimidine dimers in genomic and minichromosome DNA of yeast. We thank Dr. Wei Xiao for providingmag1, rad1, and mag1 rad1strains, and Dr. Louise Prakash for providing strain Y452. We also thank members of the Smerdon laboratory for critical discussions and technical help.

Measurement of DNA damage and repair at the nucleotide level in intact cells has provided compelling evidence for the molecular details of these events as they occur in intact organisms. Furthermore, these measurements give the most accurate picture of the rates of repair in different structural domains of DNA in chromatin. In this report, we describe two methods currently used in our laboratories to map DNA lesions at (or near) nucleotide resolution in yeast cells. The low-resolution method couples damage-specific strand breaks in DNA with indirect end-labeling to measure DNA lesions over a span of 1.5 to 2 kb of DNA sequence. The resolution of this method is limited by the resolution of DNA length measurements on alkaline agarose gels (about +/-20 bp on average). The high-resolution method uses streptavidin magnetic beads and special biotinylated oligonucleotides to facilitate end-labeling of DNA fragments specifically cleaved at damage sites. The latter method maps DNA damage sites at nucleotide resolution over a shorter distance (<500 bp), and is constrained to the length of DNA resolvable on DNA sequencing gels. These methods are used in tandem for answering questions regarding DNA damage and repair in different chromatin domains and states of gene expression.

Rpb9, a nonessential subunit of RNA polymerase II (Pol II), has multiple transcription-related functions in Saccharomyces cerevisiae, including transcription elongation and transcription-coupled repair (TCR).Here we show that, in response to UV radiation, Rpb9 also functions in promoting ubiquitylation and degradation of Rpb1, the largest subunit of Pol II.This function of Rpb9 is not affected by any pathways of nucleotide excision repair, including TCR mediated by Rpb9 itself and by Rad26.Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker.The Zn2 domain, which is dispensable for transcription elongation and TCR functions, is essential for Rpb9 to promote Rpb1 degradation, whereas the Zn1 and linker domains, which are essential for transcription elongation and TCR functions, play a subsidiary role in Rpb1 degradation.Coimmunoprecipitation analysis suggests that almost the full length of Rpb9 is required for a strong interaction with the core Pol II: deletion of the Zn2 domain causes dramatically weakened interaction, whereas deletion of Zn1 and the linker resulted in undetectable interaction.Furthermore, we show that Rpb1, rather than the whole Pol II complex, is degraded in response to UV radiation and that the degradation is primarily mediated by the 26S proteasome.

Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR.

Electroacupuncture (EA) has been demonstrated to promote the functional recovery of neurons following spinal cord injury (SCI); however, the mechanisms underlying its effects have yet to be elucidated. The Wnt/β-catenin signaling pathway has been implicated in the regulation of the balance between growth, proliferation and differentiation of neural precursor cells. The present study aimed to investigate the effects of EA therapy on Wnt/β‑catenin‑regulated gene expression and neuronal recovery in rats with SCI. The Allen method was used to establish SCI in rats, and alterations in Wnt1 and Nestin mRNA and protein expression levels in response to SCI were determined on days 1, 3, 7 and 14 post‑injury using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. To evaluate the effects of EA treatment on SCI, the following four treatment groups were employed: SCI, SCI + EA, SCI + lithium chloride (LiCl) and SCI + LiCl + EA. The protein expression levels of Wnt1, Nestin and nuclear β‑catenin were evaluated on day 3 post‑treatment, and neuronal nuclear antigen (NeuN) protein expression levels were evaluated on day 21 post‑treatment using western blot analysis. The Basso, Beattie and Bresnahan scoring method was used to evaluate spinal cord recovery on day 28 post‑treatment across the four treatment groups. EA therapy at the Dazhui and Mingmen acupuncture points significantly increased the expression levels of Wnt1, Nestin, β‑catenin and NeuN, thus suggesting that EA therapy may promote spinal cord recovery following injury. The underlying mechanism was demonstrated to involve enhanced Wnt/β‑catenin signaling, which may promote the proliferation and differentiation of neural stem cells. However, further studies are required to elucidate the detailed effects and underlying molecular mechanisms of EA therapy on SCI.

Patients with obstructive sleep apnea syndrome (OSAS) showed higher prevalence in cardiovascular diseases due to aberrant hypoxia and oxidative stress. However, not all OSAS patients end up with cardiovascular disorders, and identification of novel biomarker will be invaluable for differentiating patients at risk. Here we tested the serum matrix metalloproteinase-9 (MMP-9) levels in 47 untreated OSAS patients and found that the MMP-9 level was positively correlated with severity of OSAS, which was consistent with hypoxia degree and duration. Besides, the MMP-9 level was higher in patients complicated with systematic hypertension (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mrow><mml:mi>P</mml:mi></mml:mrow></mml:math> &lt; 0.001). Furthermore, we selected those OSAS patients without any cardiovascular dysfunction (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M2"><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">35</mml:mn></mml:math>) and followed up for up to five years. By the end of follow-up, 12 patients had hypertension onset and 3 patients had left ventricular hypertrophy. By analyzing the clinical outcomes with MMP-9 expression, we demonstrated that high serum MMP-9 in OSAS patients was a risk factor for occurrence of cardiovascular diseases. In addition, we cultured the vascular endothelial cells (VEC) from rat aorta in hypoxia condition to investigate whether MMP-9 was elevated due to hypoxia in OSAS patients. Cellular results revealed that the expression, secretion, and activity of MMP-9 were all upregulated by hypoxia and can cleave the beta2-adrenergic receptor ( β 2AR) on VEC surface. Our results not only determined MMP-9 as a risk factor for cardiovascular diseases in OSAS patients, but also showed the possible involvement of hypoxia-MMP-9- β 2AR signaling axis.

Shewanella oneidensis is an extensively studied bacterium capable of respiring minerals, including a variety of iron ores, as terminal electron acceptors (EAs). Although iron plays an essential and special role in iron respiration of S. oneidensis, little has been done to date to investigate the characteristics of iron transport in this bacterium. In this study, we found that all proteins encoded by the pub-putA-putB cluster for putrebactin (S. oneidensis native siderophore) synthesis (PubABC), recognition-transport of Fe3+-putrebactin across the outer membrane (PutA), and reduction of ferric putrebactin (PutB) are essential to putrebactin-mediated iron uptake. Although homologs of PutA are many, none can function as its replacement, but some are able to work with other bacterial siderophores. We then showed that Fe2+-specific Feo is the other primary iron uptake system, based on the synthetical lethal phenotype resulting from the loss of both iron uptake routes. The role of the Feo system in iron uptake appears to be more critical, as growth is significantly impaired by the absence of the system but not of putrebactin. Furthermore, we demonstrate that hydroxyl acids, especially α-types such as lactate, promote iron uptake in a Feo-dependent manner. Overall, our findings underscore the importance of the ferrous iron uptake system in metal-reducing bacteria, providing an insight into iron homeostasis by linking these two biological processes.IMPORTANCES. oneidensis is among the first- and the best-studied metal-reducing bacteria, with great potential in bioremediation and biotechnology. However, many questions regarding mechanisms closely associated with those applications, such as iron homeostasis, including iron uptake, export, and regulation, remain to be addressed. Here we show that Feo is a primary player in iron uptake in addition to the siderophore-dependent route. The investigation also resolved a few puzzles regarding the unexpected phenotypes of the putA mutant and lactate-dependent iron uptake. By elucidating the physiological roles of these two important iron uptake systems, this work revealed the breadth of the impacts of iron uptake systems on the biological processes.

Cisplatin-based chemotherapeutic regimens are frequently used for treatments of solid tumors. However, tumor cells may have inherent or acquired cisplatin resistance, and the underlying mechanisms are largely unknown. We performed genome-wide screening of genes implicated in cisplatin resistance in A375 human melanoma cells. A substantial fraction of genes whose disruptions cause cisplatin sensitivity or resistance overlap with those whose disruptions lead to increased or decreased cell growth, respectively. Protein translation, mitochondrial respiratory chain complex assembly, signal recognition particle-dependent cotranslational protein targeting to membrane, and mRNA catabolic processes are the top biologic processes responsible for cisplatin sensitivity. In contrast, proteasome-mediated ubiquitin-dependent protein catabolic process, negative regulations of cellular catabolic process, and regulation of cellular protein localization are the top biologic processes responsible for cisplatin resistance. ZNRF3, a ubiquitin ligase known to be a target and negative feedback regulator of Wnt-β-catenin signaling, enhances cisplatin resistance in normal and melanoma cells independently of β-catenin. Ariadne-1 homolog (ARIH1), another ubiquitin ligase, also enhances cisplatin resistance in normal and melanoma cells. By regulating ARIH1, neurofibromin 2, a tumor suppressor, enhances cisplatin resistance in melanoma but not normal cells. Our results shed new lights on cisplatin resistance mechanisms and may be useful for development of cisplatin-related treatment strategies.-Ko, T., Li, S. Genome-wide screening identifies novel genes and biological processes implicated in cisplatin resistance.

Abstract Doping of materials beyond the dopant solubility limit remains a challenge, especially when spatially nonuniform doping is required. In 2D materials with a high surface‐to‐volume ratio, such as transition metal dichalcogenides, various post‐synthesis approaches to doping have been demonstrated, but full control over spatial distribution of dopants remains a challenge. A post‐growth doping of single layers of WSe 2 is performed by adding transition metal (TM) atoms in a two‐step process, which includes annealing followed by deposition of dopants together with Se or S. The Ti, V, Cr, and Fe impurities at W sites are identified by using transmission electron microscopy and electron energy loss spectroscopy. Remarkably, an extremely high density (6.4–15%) of various types of impurity atoms is achieved. The dopants are revealed to be largely confined within nanostripes embedded in the otherwise pristine WSe 2 . Density functional theory calculations show that the dislocations assist the incorporation of the dopant during their climb and give rise to stripes of TM dopant atoms. This work demonstrates a possible spatially controllable doping strategy to achieve the desired local electronic, magnetic, and optical properties in 2D materials.

Abstract Exciton–polariton coupling between transition metal dichalcogenide (TMD) monolayer and plasmonic nanostructures generates additional states that are rich in physics, gaining significant attention in recent years. In exciton–polariton coupling, the understanding of electronic-energy exchange in Rabi splitting is critical. The typical structures that have been adopted to study the coupling are “TMD monolayers embedded in a metallic-nanoparticle-on-mirror (NPoM) system.” However, the exciton orientations are not parallel to the induced dipole direction of the NPoM system, which leads to inefficient coupling. Our proposed one-dimensional plasmonic nanogrooves (NGs) can align the MoS 2 monolayers’ exciton orientation and plasmon polaritons in parallel, which addresses the aforementioned issue. In addition, we clearly reveal the maximum surface potential (SP) change on intermediate coupled sample by the photo-excitation caused by the carrier rearrangement. As a result, a significant Rabi splitting (65 meV) at room temperature is demonstrated. Furthermore, we attribute the photoluminescence enhancement to the parallel exciton–polariton interactions.

All-optical control of nonlinear photonic processes in nanomaterials is of significant interest from a fundamental viewpoint and with regard to applications ranging from ultrafast data processing to spectroscopy and quantum technology. However, these applications rely on a high degree of control over the nonlinear response, which still remains elusive. Here, we demonstrate giant and broadband all-optical ultrafast modulation of second-harmonic generation (SHG) in monolayer transition-metal dichalcogenides mediated by the modified excitonic oscillation strength produced upon optical pumping. We reveal a dominant role of dark excitons to enhance SHG by up to a factor of ∼386 at room temperature, 2 orders of magnitude larger than the current state-of-the-art all-optical modulation results. The amplitude and sign of the observed SHG modulation can be adjusted over a broad spectral range spanning a few electronvolts with ultrafast response down to the sub-picosecond scale via different carrier dynamics. Our results not only introduce an efficient method to study intriguing exciton dynamics, but also reveal a new mechanism involving dark excitons to regulate all-optical nonlinear photonics.

Many ion channels are known to behave as an allosteric protein, coupling environmental stimuli captured by specialized sensing domains to the opening of a central pore. The classic Monod-Wyman-Changeux (MWC) model, originally proposed to describe binding of gas molecules to hemoglobin, has been widely used as a framework for analyzing ion channel gating. Here we address the issue of how accurate the MWC model predicts activation of the capsaicin receptor TRPV1 by vanilloids. Taking advantage of a concatemeric design that makes it possible to lock TRPV1 in states with zero-to-four bound vanilloid molecules, we showed quantitatively that the overall gating behavior is satisfactorily predicted by the MWC model. There is however a small yet detectable subunit position effect: ligand binding to two kitty-corner subunits is 0.3-to-0.4 kcal/mol more effective in inducing opening than binding to two neighbor subunits. This difference-less than 10% of the overall energetic contribution from ligand binding-might be due to the restriction on subunit arrangement imposed by the planar membrane; if this is the case, the position effect is not expected in hemoglobin, in which each subunit is related equivalently to all the other subunits.

Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. Rad26, a member of the Swi2/Snf2 superfamily of proteins, has been shown to be involved in a specialized NER process called transcription coupled NER. Rad16, another member of the same protein superfamily, has been shown to be required for genome-wide NER. Here we show that Rad16 and Rad26 play different roles in repairing repressed and actively transcribed genes in yeast. Rad16 is partially dispensable, and Rad26 plays a significant role in repairing certain regions of the repressed GAL1-10, PHO5 and ADH2 genes, especially in the core DNA of well-positioned nucleosomes. Simultaneous elimination of Rad16 and Rad26 results in no detectable repair in these regions of the repressed genes. Transcriptional induction of the GAL1-10 genes abolishes the role of Rad26, but does not affect the role of Rad16 in repairing the nontranscribed strand of the genes. Interestingly, when the transcription activator Gal4 is eliminated from the cells, Rad16 becomes partially dispensable and Rad26 plays a significant role in repairing both strands of the GAL1-10 genes even under inducing conditions. Our results suggest that Rad16 and Rad26 play different and, to some extent, complementary roles in repairing both strands of repressed genes, although the relative contributions of the two proteins can be different from gene to gene, and from region to region of a gene. However, Rad16 is solely responsible for repairing the nontranscribed strand of actively transcribed genes.

Previous reports show that SIRT6 serves as a critical modulator of the development of multiple malignancies as well as other disorders. However, its role in nasopharyngeal carcinoma (NPC) is unknown. Thus, we elucidated the effects of SIRT6 on the survival of NPC cells, and modulation of cell death.We found that expression of SIRT6 is downregulated in ten human NPC specimens as well as in the human NPC cell lines, 5-8 F and CNE1, as compared with that in healthy tissues and normal nasopharyngeal NP69 cells. The MTT assay and colony formation assay revealed that upregulation of SIRT6 impaired the proliferation, as well as the survival of 5-8 F and CNE1 cells. The TUNEL assay, annexin V-FITC/propidium iodide, and flow cytometry were performed to detect apoptosis. The results revealed that the expression of SIRT6 resulted in increased apoptosis.Western blotting results showed that SIRT6 overexpression decreased anti-apoptotic Bcl-2 levels, whereas it promoted an increase in pro-apoptotic Bax and cleaved caspase-3 levels. Moreover, NF-κB levels were markedly reduced in cells expressing SIRT6, whereas they were increased in cells transfected with shRNA-SIRT6. Recovery of NF-κB expression was found to counter the suppressive influence of SIRT6 on NPC cell survival, whereas, NF-κB knockdown increased apoptosis of NPC cells.Thus, the findings of our study offer insight into the biological and molecular mechanisms underlying the development of NPC and may lead to the development of new and innovative strategies for the treatment of NPC.

Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus.Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets.A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.

Clubroot caused by Plasmodiophora brassicae is a severe threat to the production of Brassica napus, worldwide. The cultivation of resistant varieties is the most efficient and environmentally friendly way to limit disease spread. We developed a highly resistant B. napus line, ZHE226, containing the resistance locus PbBa8.1. However, ZHE226 seeds contain high erucic acid content, which limits its cultivation owing to its low edible oil quality. A segregation population of BC3F2 was developed by crossing ECD04, a resistant European turnip donor, with Huangshuang5, an elite variety with no erucic acid in its seeds, as a recurrent plant. Fine mapping using the bulk segregation analysis sequencing (BSA-Seq) approach detected PbBa8.1 within a 2.9 MB region on chromosome A08. Interestingly, the previously reported resistance gene Crr1a was found in the same region. Genetic analysis revealed that the CAP-134 marker for Crr1a was closely linked with clubroot resistance (CR). Thus, PbBa8.1 and Crr1a might be allelic for CR. Moreover, comparative and genetic analysis showed that high erucic acid in the seeds of ZHE226 was due to linkage drag of fatty acid elongase 1 (FAE1) in the ECD04 line, which was located in the interval of PbBa8.1 with a physical and genetic distance of 729 Kb and 1.86 cm, respectively. Finally, a clubroot-resistant line with a low erucic acid content was successfully developed through gene-specific molecular marker assistant selection from BC4F4. These results will accelerate CR breeding programs in B. napus.

Abstract Mixed‐dimensional heterostructures which combine materials with different dimensions have emerged to expand the scope and functionality of van der Waals heterostructures. Here, a direct synthesis method of molybdenum disulfide/double‐wall carbon nanotube (MoS 2 /DWCNT) mixed‐dimensional heterostructures by sulfurating a molten salt, Na 2 MoO 4 , on a substrate covered with a DWCNT film is reported. The synthesized heterostructures are comprehensively characterized and their stacking order is confirmed to be MoS 2 under the DWCNTs, although the DWCNT film is transferred on the substrate first. Moreover, field‐effect transistors based on the heterostructure are fabricated for photodetection, and an abnormal negative photoresponse is discovered due to the strong carrier transfer in the mixed‐dimensional heterostructures under light incidence. The MoS 2 /DWCNT heterostructure results provide a new approach for the synthesis and applications of mixed‐dimensional heterostructures.