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Due to the inherent volume expansion effect and low conductivity of silicon monoxide (SiOx, 0

Ni-rich layered structure materials are appealing cathodes for high-energy-density lithium-ion batteries developed for electric vehicles, drones, power tools, etc. However, poor interfacial stability between a Ni-rich cathode and carbonate electrolyte, especially at high temperatures, and fast capacity fading still hinder their mass market penetration. Here, we investigate cyclopentyl isocyanate (CPI) with a single isocyanate (-NCO) functional group as a bifunctional electrolyte additive for the first time to improve the interfacial stability of Ni-rich cathode LiNi0.83Co0.12Mn0.05O2 (NCM83). With an electrolyte containing 2 wt % CPI, the NCM83 cathode shows capacity retention of up to 92.3% after 200 cycles at 1C and 30 °C, much higher than that with the standard electrolyte (78.6%). It is demonstrated that the -NCO of CPI could largely inhibit the thermal decomposition of LiPF6 salt and scavenge water and hydrogen fluoride (HF) species, improving electrolyte stability. More importantly, the additive CPI could be preferentially oxidized, forming a stabilized and protective cathode electrolyte interphase (CEI) layer on the surface of NCM83, which effectively suppresses the parasitic side reactions and maintains the superior interfacial charge-transfer and lithium-ion diffusion kinetics. Both functions enable a significant improvement in electrochemical performance at both 30 and 60 °C.

In recent years, with the rapid developments of high-technology industries such as information technology and electric vehicles, there are urgent need to develop new generations of lithium-ion batteries with higher energy density, longer cycle life and improved safety. In addition to the development of high specific energy cathode and high specific capacity anode materials, regulating the stability of the electrode/electrolyte interface is critical to achieve and balance various performances of the batteries and finally realize their commercial application widely. However, the traditional carbonate electrolytes not only suffer from severe oxidation decomposition when the charging voltage is higher than 4.3 V (vs. Li/Li+), but also show poor compatibility with high-capacity silicon or silicon-carbon (Si-C) composite anodes and lithium metal anodes. Moreover, the traditional carbonate electrolytes are flammable, which is still a big safety concern to address for lithium ion battery. Therefore, rational design of electrolytes that match the high voltage cathode, high specific capacity Si-C abode or lithium metal anode, and has better safety property and especially under harsh conditions, has become a decisive factor for the rapid developments of high specific energy lithium-ion batteries. The present work reviews different kinds of liquid electrolytes with functional additives and utilization of solid state electrolytes which are explored by our group in the past 15 years. The design strategies and systematic investigations of the electrolytes recipes include: (1) Developing anti-oxidation solvent systems, for example, applications of new high-voltage nitriles or fluorinated solvents for high-voltage cathodes, such as suberonitrile (SUN), fluoroethylene carbonate (FEC) and ethyl-(2,2,2-trifluoroethyl) carbonate (ETFEC); (2) exploiting some novel multifunctional solvents or additives which contain flame retardant groups or can be used as film-forming agents for high-voltage cathodes. For example, some P-containing compounds such as N , N -diallyl-diethoxy phosphoramide (DADEPA), phenoxy cyclophosphazene (PFPN), and ionic liquid such as N-methyl-N-butylpyrrolidinium bis(trifluoromethylsulfonyl)imide (Py14TFSI), etc. In addition, we have also investigated a series of solid electrolytes such as Garnet-type electrolytes, gel polymer or composite polymer electrolyte with inorganic additives, which could greatly reduce the safety risk of batteries. These works include measurements of the activation energy and ion transport mechanism of oxide solid electrolyte with solid NMR techniques, interface modification of sulfide-type solid state battery with ionic liquids, and interface passivation mechanism of PEO-based polymer electrolyte, etc. Finally, some discussions and future perspectives in developing high-voltage and highly-safe liquid electrolyte and flexible solid-state electrolytes for all solid state batteries are presented. Although the commonly used high-voltage electrolytes are mainly composed of lithium salts, anti-oxidation solvents and functional additives such as nitriles, sulfones, fluoro-ethers or fluoro-carbonates, However, the acting mechanisms of those high-voltage electrolytes, especially solvents, salts and additives, are still not clear, especially the composition, structure and evolution of the electrochemical interfaces are lack of quantitative understanding. Therefore, combining theoretical calculation and advanced in-/ex-situ interface characterization techniques, fully understanding the working mechanism of the additives in high-voltage systems and the effective components of interface film, and designing novel functional electrolyte additives are crucial for the development of a new generation of high-specific energy lithium batteries. In addition, applications of flame-retardant solvents or additives to develop flame-retardant liquid-type electrolytes and developing new solid state electrolytes are both important strategies to reduce the potential safety hazards of lithium ion batteries. The future research should focus on how to improve the compatibility between flame-retardant solvents/additives and the cathode/anode interfaces in liquid electrolytes, minimize the negative impact on battery performance (cycle performance, rate performance, etc.), enhance the ionic conductivity and mechanical flexibilities of the solid electrolytes, optimize the electrolyte phase structure/mechanical properties, and regulate the stability of interfaces to ensure the long-term cycling stability of lithium metal anodes and metal oxide cathodes.

α-Terpineol, terpinen-4-ol, and δ-terpineol, isomers of terpineol, are among the compounds that give Cinnamomum longepaniculatum leaf oil its distinguished pleasant smell. The objective of this study was to evaluate the antimicrobial activity of these three isomeric terpineols. The determination of antibacterial activity was based on the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC). Changes in time-kill curve, alkaline phosphatase (AKP), UV-absorbing material, membrane potential, and scanning electron microscopy (SEM) were measured to elucidate the possible antimicrobial mechanism. α-Terpineol, terpinen-4-ol, and δ-terpineol demonstrated good inhibitory effects against several gram-negative bacteria, particularly Shigella flexneri. MIC and MBC of α-terpineol and terpinen-4-ol were similar (0.766 mg/mL and 1.531 mg/mL, respectively) for S. flexneri, while the MIC and MBC values of δ-terpineol were 0.780 mg/mL and 3.125 mg/mL, respectively. Time-kill curves showed that the antibacterial activities of the tested compounds were in a concentration-dependent manner. Release of nucleic acids and proteins along with a decrease in membrane potential proved that α-terpineol, terpinen-4-ol, and δ-terpineol could increase the membrane permeability of Shigella flexneri. Additionally, the release of AKP suggested that the cell wall was destroyed. SEM analysis further confirmed that S. flexneri cell membranes were damaged by α-terpineol, terpinen-4-ol, and δ-terpineol. Our research suggests that these three isomeric terpineols have the potential of being used as natural antibacterial agents by destroying the cell membrane and wall, resulting in cell death. However, the specific antibacterial activity differences need further investigation.

Polysaccharide derived from natural products has a wide range of sources and mild properties, and exhibit various bioactivities. Ascorbic acid is one of the most important nutrients in fruits and vegetables, as well as their products. Ascorbic acid and polysaccharide coexist in many systems during food production and processing. Many studies have found that ascorbic acid at low concentrations degrades polysaccharide derived from natural products via hydroxyl radical. In this paper, the research progress on ascorbic acid induced polysaccharide degradation is summarized from four aspects: mechanism of action, analytical methods, influencing factors and bioactivity of degradation products. It is expected to provide a theoretical basis for further research.

A protocol of visible-light-promoted C2 selective arylation of quinoline and pyridine N-oxides, with diaryliodonium tetrafluoroborate as an arylation reagent, using eosin Y as a photocatalyst for the construction of N-heterobiaryls was presented. This methodology provided an efficient way for the synthesis of 2-aryl-substituted quinoline and pyridine N-oxides. This strategy has the following advantages: specific regioselectivity, simple operation, good functional group tolerance, and high to moderate yields under mild conditions.

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+/ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid‐induced necrotic death of cells including SH‐SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site‐specific phosphorylation inactivates PARP1, inhibiting ionophore‐induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR‐mediated anti‐necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling.

An atom-economical oxovanadate-catalysed cycloisomerization of hydroxy enynes for the synthesis of bicyclo[4.3.0]non-1(9)-en-2-ones is disclosed, which can be rationalised through a cascade reaction of a dissociative Meyer–Schuster rearrangement to allenyl vanadates, followed by a thermal intramolecular Diels–Alder (IMDA) reaction and hydrolytic regeneration of the catalyst.

The natural deep eutectic solvent (NADES) is an excellent solvent for insoluble natural products and medicines. Eutectogels formed by gelation of NADESs are interesting materials that deserve attention. In this study, xanthan gum was used as a gelator to gel choline chloride-xylitol with different water contents in virtue of the excellent solubility of choline chloride-xylitol (1:1) to quercetin. We observed that water was critical to the formation of eutectogels. An MTT assay indicated that our eutectogel had excellent biocompatibility as its corresponding hydrogel. According to rheological tests, xanthan gum-based eutectogels had better viscoelastic properties, higher thermal stability, and more defined shear thinning behavior than its corresponding hydrogel. Texture profile analysis showed that eutectogels with less water content had higher hardness and adhesiveness. Meanwhile, Differential scanning calorimeter (DSC) results suggested that the various rheological and texture properties of eutectogels could be attributed to changes in the water state, which was influenced by the hydrogen bonding network of NADES. This biocompatible eutectogel with tunable properties was expected to find applications in novel drug delivery vehicles, which are widely used in the fields of medicine and food.

A stabilized cathode electrolyte interphase (CEI) formed on the cathode electrode by virtue of effective electrolyte additives is crucial for prolonging the lifespan of lithium-ion battery adopting high energy density Ni-rich cathode materials. Here, we report a novel multifunctional additive trimethylsilyl 2-(fluorosulfonyl)difluoroacetate (TMSFS) for LiNi0.9Co0.05Mn0.05O2 (NCM90). By adding 0.5% TMSFS, the NCM90 electrode shows an enhanced capacity retention of 85.1% at 1C up to 200 cycles, superior over that of 73.1% for baseline electrolyte. The improvement on cycling performance can be also achieved at harsh testing conditions (e.g., 60 °C and even a high voltage of 4.4 V), along with fast electrode kinetics in the presence of TMSFS. It's demonstrated that TMSFS additive could scavenge the undesired H2O, PF5 and HF species in electrolyte, alleviating the subsequent damages to both cathode-electrolyte interface and NCM90 cathode structure. More importantly, a compact, inorganics-enriched CEI layer formed in TMSFS-added electrolyte is further revealed, rationalizing the impressive improvement in electrochemical performance. Furthremore, we also prove the effectiveness of TMSFS additive for graphite||NCM90 full cells.

The water-extracted polysaccharide from Portulaca oleracea L. mainly consisted of two pectic polysaccharides designated as POWP-H and POWP-L. The POWP-L was rich in linear homogalacturonan with GalA content of 77.6%, while POWP-H might be a rhamnogalacturonan I (RG-I) pectin composed of Rha (15.7%), Ara (31.7%), Gal (42.5%) and GalA (10.1%). The structure of POWP-H was elucidated by partial degradation, methylation analysis combined with GC-MS method and NMR spectroscopy. It had a weight-average molecular weight of 1.2 × 106 g/mol and high degree of branch of 0.57. The presence of abundant arabinogalactan-II and certain galactan side chains in POWP-H was verified by the T-Araf (17.4%), 1,3,6-Galp (11.8%), 1,3-Galp (8.1%), T-Galp (13.4%) and 1,4-Galp (14.3%). The evidence for the RG-I backbone of POWP-H was demonstrated by the structure analysis of its degraded product POWP-H-H, which had the backbone of →4)-α-GalpA-(1 → 2)-α-Rhap-(1 → 4)-α-GalpA-(1 → 2,4)-α-Rhap-(1→ substituted by short side chain of β-Galp-(1 → 4)-β-Galp-(1→ at O-4 position of →2,4)-α-Rhap-(1 → . Moreover, the POWP-H-H was mainly O-acetylated at O-3 position of α-Rhap with 24.8% acetylation degree. In conclusion, POWP-H was mainly the highly branched and acetylated RG-I pectin with relative short RG-I backbone and abundant arabinogalactan II and certain galactan side chains. Besides, it adopted a flexible chain conformation in 0.1 M NaNO3 solution.

β-Glucans are widely found in plants and microorganisms, which has a variety of functional activities. During production and application, interactions with other components have a great influence on the structure and functional properties of β-glucan. In this paper, interactions (including non-covalent interaction and free-radical reaction) between natural product derived β-glucan and ascorbic acid, polyphenols, bile acids/salts, metal ion or other compounds were summarized. Besides, the mechanism and influence factors of interactions between β-glucan and small-molecule compounds, and their effects on the functional properties of β-glucan were detailed. This review aims to develop an understanding and practical suggestions on interactions between β-glucan and small-molecule compounds, which is expected to provide a useful reference for processing and application.

In this paper, the polystyrene–silica hybrid particles were prepared by mechanochemical method. The surface activity index of the hybrid particle was prepared by mechanochemical method, and the contact angle is 128.8°, which indicates that it has the very good hydrophobicity. We can observe that the surface of the modified SiO2 coated with a layer of polystyrene under the transmission electron microscopy. In infrared, we found that there are Si–C bonds on the surface of the modified SiO2, which indicates that styrene monomer is successfully grafted to the surface of silica in the form of polystyrene whose maximum viscosity average degree of polymerization is 158 under mechanical force. The grafting rate was 4.28% measured by thermogravimetric analysis.

To elucidate the inhibitory effects of different herbicides on soil nitrification, eight widely used herbicides, i.e., acetochlor, atrazine, dicamba, isoproturon, paraquat, puma, tribenuron-methyl, and 2,4-dichlorophenoxyacetic acid butyl ester (2,4-Dbe), which represent different chemical taxonomy were selected. Our results indicated that herbicide 2,4-Dbe displayed the best inhibitory effect on nitrification, followed by puma and tribenuron-methyl, whereas the remaining five herbicides exhibited less effect when 10 mg of active ingredient (A.I.) of every herbicide per kg of soil was applied in vegetable-planting soil. The inhibition appeared when 5–100 mg of A.I. 2,4-Dbe was employed, which was enhanced with an increment in its dose in both vegetable-planting and fluvo-aquic soils. However, the inhibitory effect of 10 mg of A. I. 2,4-Dbe exhibited obvious differences in these two types of soils, where the duration of inhibition was shorter as it only continued about a week in fluvo-aquic and calcic cambisols soils with strong nitrification activity but poorer effect as compared to 10 mg of dicyandiamide (DCD). In contrast, the duration of inhibition exceeded 2 months in dryland red and shajiang black soils with a weak nitrification activity which was equivalent to DCD. In addition, comparing with five nitrification inhibitors, 10 mg of 2,4-Dbe had better inhibition than the substituted pyrimidine (AM) and sulfocarbamide (SU), but was equivalent to DCD, nitrapirin, and 3,4-dimethylpyrazole phosphate (DMPP) at their recommended application rates in dryland red soil. These obtained data clearly indicated that 2,4-Dbe could play a stronger role as a nitrification inhibitor in soils.

A series of silicon-containing derivatives of linear alicyclic musks comprising sila-Rosamusk (3b), sila-Romandolide (5b), sila-Applelide (11b), and the corresponding dehydro derivatives was synthesized from sila-analogs of Artemone and Herbac, respectively, by means of sodium borohydride reduction and subsequent esterification with the corresponding acid chlorides in the presence of triethylamine. The olfactory properties of the new sila-odorants 3b–13b are reported in comparison with their carba-analogs 3a–13a, and quantitative threshold data allowed the generation of an improved musk olfactophore model featuring a correlation of 80.5 % with a null-cost distance of 244. This olfactophore model shows that it is likely that linear and macrocyclic musks address the same odorant receptors, and it should facilitate the design of new musks.

A novel synthetic approach to (±)- Z -recifeiolide 6, a 12-membered-ring lactone which can be selectively isomerized into ( E )-recifeiolide, a natural antibiotic product isolated from fungus ( Cephalosporium recifei ) is reported. The synthesis is accomplished in five steps starting from readily available cyclooctanone and acetaldehyde based on the Lewis acid-catalyzed TMS-directed oxy-2-oxonia-Cope rearrangement. The work represents a novel strategy to assemble related macrolides.

Lactic acid has been used as a bio-based green solvent to study the ultrasound-assisted scale-up synthesis. We report here, for the first time, on the novel and scalable process for synthesis of pyrrole derivatives in lactic acid solvent under ultrasonic radiation. Eighteen pyrrole derivatives have been synthesized in lactic acid solvent under ultrasonic radiation and characterized by (1)H NMR, IR, ESI MS. The results show, under ultrasonic radiation, lactic acid solvent can overcome the scale-up challenges and exhibited many advantages, such as bio-based origin, shorter reaction time, lower volatility, higher yields, and ease of isolating the products.

The construction and application of a new type of composite material are achieved more and more attention. However, expected Sb2Se3/attapulgite composites aim to use the low price, and high adsorption of attapulgite in assembling Sb2Se3 is quite difficult to be acquired by a facile and benign environmental hydrothermal method. In this manuscript, we developed a new way for preparation of an emerging composite by means of Sb2O3 as a media linking Sb2Se3 and attapulgite together, and finally won an emerging composite Sb2Se3/Sb2O3@attapulgite, which presented an excellent catalytic properties for catalytic hydrogenation of p-nitrophenol. It was noted that the Sb2Se3/Sb2O3@attapulgite composites exhibited a high conversion rate for the hydrogenation of p-nitrophenol that was up to 90.7% within 15 min, which was far more than the 61.5% of Sb2Se3 sample. The excellent catalytic performance was attributed to the highly dispersion Sb2Se3 microbelts and Sb2Se3@Sb2O3@attapulgite rods, which would improve the adsorption of the reactant species and facility electronic transfer process of the catalytic hydrogenation of p-nitrophenol.

The sensitivity of Xeroderma pigmentosa (XP) patients to sunlight has spurred the discovery and genetic and biochemical analysis of the eight XP gene products (XPA-XPG plus XPV) responsible for this condition. These studies also have served to elucidate the nucleotide excision repair (NER) process, especially the critical role played by the XPA protein. More recent studies have shown that NER also involves numerous other proteins normally employed in DNA metabolism and cell cycle regulation. Central among these is ataxia telangiectasia and Rad3-related (ATR), a protein kinase involved in intracellular signaling in response to DNA damage, especially replicative and transcription stresses. This review summarizes recent findings on the interplay between ATR as a DNA damage signaling kinase and as a novel ligand for intrinsic cell death proteins to delay damage-induced apoptosis, and on ATR’s regulation of XPA and the NER process for repair of UV-induced DNA adducts. ATR’s regulatory role in the cytosolic-to-nuclear translocation of XPA will be discussed. In addition, recent findings elucidating a non-NER role for XPA in DNA metabolism and genome stabilization at ds-ssDNA junctions, as exemplified in prematurely aging progeroid cells, also will be reviewed.

A sequential Diels-Alder reaction/rearrangement sequence was developed for the synthesis of diverse functionalized bicyclo[2.2.1]heptanes as novel floral and woody odorants. The outcome of the rearrangement depended on the substitution pattern of the dienes. 2D NMR analysis has established the correct relative configuration of the bicyclo[2.2.1]heptanone, which was originally misassigned. Furthermore, when the initiating DA reaction was catalyzed by a chiral Lewis acid, the bicyclo[2.2.1]heptane derivatives including (+)-herbanone can be obtained in an enantiomeric ratio (er) up to 96.5:3.5.

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a -G*CN- sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of ‘stacked’ (S) and ‘B-type’ (B) conformers, whereas the N-acetylated FAAF also samples a ‘wedge’ (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A- to -T polarity swap in the G*CA/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2- to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3′-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli.

Lowering the driving voltage of organic light-emitting diodes (OLEDs) is an important approach to reduce their energy consumption. We have fabricated a series of bifunctional devices (OLEDs and photovoltaics) using rubrene and fullerene (C60) as the active layer, in which the electroluminescence threshold voltage(~1.1 V) was half the value of the bandgap of rubrene. Magneto-electroluminescence (MEL) response of planner heterojunction diodes exhibited a small increase in response to a low magnetic field strength (20 mT). When a hole-transport layer with a low mobility was included in these devices, the MEL response reversed in shape and simultaneously, the EL threshold voltage became larger than the bandgap voltage. When bulk heterojunction device was examined, the amplitude of MEL curves presented an anomalous voltage-dependence. Following an analysis of the MEL responses of these devices, we proposed that the EL of half-bandgap-voltage device originated from bimolecular triplet-triplet annihilation in the rubrene film, rather than from singlet excitons that formed via an interface auger recombination. This work provides critical insight into the mechanisms of OLED emission and will help advance the applications of bifunctional devices.

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

Hexavalent chromium [Cr(VI)], which is used for various industrial applications, such as leather tanning and chroming, can cause a number of human diseases including inflammation and cancer. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study provides evidence that Cr(VI) enhances reactive oxygen species (ROS) accumulation by inhibiting the mitochondrial respiratory chain complex (MRCC) I. Cr(VI) did not affect the expression levels of antioxidative proteins such as superoxide dismutase (SOD), catalase and thioredoxin (Trx), indicating that the antioxidative system was not involved in Cr(VI)-induced ROS accumulation. We found that ROS mediated caspase-3 activation partially depends on the downregulation of the heat shock protein (HSP) 70 and 90. In order to confirm our hypothesis that ROS plays a key role in Cr(VI)-mediated cytotoxicity, we used N-acetylcysteine (NAC) to inhibit the accumulation of ROS. NAC successfully blocked the inhibition of HSP70 and HSP90 as well as the activation of caspase-3, suggesting that ROS is essential in Cr(VI)-induced caspase-3 activation. By applying different MRCC substrates as electron donors, we also confirmed that Cr(VI) could accept the electrons leaked from MRCC I and the reduction occurs at MRCC I. In conclusion, the present study demonstrates that Cr(VI) induces ROS-dependent caspase-3 activation by inhibiting MRCC I activity, and MRCC I has been identified as a new target and a new mechanism for the apoptosis-inducing activity displayed by Cr(VI).

The bending mechanical properties of bamboo plywood which was glued with phenol formaldehyde (PF) resine were detected and analysised. The study results showed that the differences of the bending strength (MOR)and the elastic modulus(MOE) of bamboo plywood prepared separately within 110-130°C were not large, and MOR and MOE exceeded all the targets ruled by JB/T 156-2004. It was proved that the property of this kind of middle temperature PF resine was very well for using in bamboo plywood. When the load was the same, the differences of the deflection、the stress of the bamboo plywood prepared on the different hot-pressing temperature were very small too, but some differences existed in the those strains，and the strains on the lower sursurface of the boards were larger than those on upper surface. When the load was smaller, The deflection、stress and strain of the boards linearly changed with the load increase, which proved that the bamboo plywood mainly produced elastic deformation under this circumstances.

2-(2′-Hydroxyphenyl)-5-amino-2H-benzotriazole was synthesized and subjected to acylamidation to yield three new ultraviolet (UV) absorbers. The largest absorption wavelength and the relationship between the concentration and the maximum absorbance of the UV absorbers were measured in dimethylsulfoxide. Then the four benzotriazole type UV absorbers were dispersed and applied onto polyester fabrics with the high temperature and pressure dyeing method. The stabilizing effect against UV light of the finished fabrics was studied by ultraviolet protection factor (UPF) and UV transmittance measurement.

Simple allenyl ethers have been shown to be efficiently activated by cationic gold(I) catalysts to form reactive dienophiles in intermolecular Diels-Alder reactions. A range of different dienes give the cycloaddition products in moderate to excellent yields and good selectivity.

Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC.

A practical procedure for palladium catalyzed Suzuki cross-coupling reaction of arylboronic acids with aryl halides, including aryl chlorides in molten tetra-n-butylammonium bromide (TBAB) was developed. The reaction exhibits high efficiency and functional group tolerance. The recovery of the catalyst and molten n-Bu4NBr was also investigated.

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.

Ionomers are co-polymers with ionic groups. One of the interesting applications of ionomer membranes is as electrolytes in proton exchange membrane (PEM) fuel cells. The most commonly used membranes in PEM fuel cells are perfluorosulfonic acid (PFSA) membranes, e.g., Nafion® from DuPontTM. Besides its dependency on temperature and hydration due to phase inversion and cluster formation, Nafion® as a polymer, exhibits strong time and rate effects. In this work, the stress–strain behavior of Nafion® at different strain rates has been obtained in an environmental chamber for various temperatures and hydrations. After a certain strain was reached in each test, stress relaxation was performed for an hour to observe the relaxation behavior of Nafion®. We attempted to use a nonlinear, time-dependent constitutive model to predict the hygro-thermomechanical behavior of Nafion®. Because a substantial component of the response is unrecoverable, a viscoplastic model was employed. The proposed two-layer viscoplasticity model consisted of an elastoplastic network that was in parallel with an elastic-viscous network (Maxwell model) which separates the rate-dependent and rate-independent behavior of the material. After obtaining the necessary parameters for different hydrations, this model showed reasonably accurate success in predicting the stress–strain behavior at different strain rates, and matched the relaxation test results. Finite element simulations based on the proposed two-layer viscoplasticity model were in good agreement with test results and can be used to study the stress–strain state of the ionomer membranes in fuel cell configurations.

A systematic spectroscopic and computational study was conducted in order to probe the influence of base sequences on stacked (S) versus B-type (B) conformational heterogeneity induced by the major dG adduct derived from the model carcinogen 7-fluoro-2-aminofluorene (FAF). We prepared and characterized eight 12-mer DNA duplexes (-AG*N- series, d[CTTCTAG*NCCTC]; -CG*N- series, d[CTTCTCG*NCCTC]), in which the central guanines (G*) were site-specifically modified with FAF with varying flanking bases (N = G, A, C, T). S/B heterogeneity was examined by CD, UV, and dynamic 19F NMR spectroscopy. All the modified duplexes studied followed a typical dynamic exchange between the S and B conformers in a sequence dependent manner. Specifically, purine bases at the 3'-flanking site promoted the S conformation (G > A > C > T). Simulation analysis showed that the S/B energy barriers were in the 14-16 kcal/mol range. The correlation times (tau = 1/kappa) were found to be in the millisecond range at 20 degrees C. The van der Waals energy force field calculations indicated the importance of the stacking interaction between the carcinogen and neighboring base pairs. Quantum mechanics calculations showed the existence of correlations between the total interaction energies (including electrostatic and solvation effects) and the S/B population ratios. The S/B equilibrium seems to modulate the efficiency of Escherichia coli UvrABC-based nucleotide excision repair in a conformation-specific manner: i.e., greater repair susceptibility for the S over B conformation and for the -AG*N- over the -CG*N- series. The results indicate a novel structure-function relationship, which provides insights into how bulky DNA adducts are accommodated by UvrABC proteins.

Currently, ionomer membranes are used in a variety of specialized applications. Such applications include, but are not limited to, dialysis, electrolysis, membrane separators, reaction catalysts and the most promising application: polymer electrolyte fuel cells. Although their use is widespread, significant gaps in understanding the mechanical behavior of these materials still remain. Many ionomer membranes change their structure, and in turn, their mechanical properties in response to applied thermal and moisture conditions that are functions of position. It has been observed that constrained materials subjected to changing environmental conditions can exhibit unusual behavior, e.g., in some cases, mechanical failure is seen in the absence of external applied mechanical loads. This condition is especially important in polymer membranes (specifically Nafion®) used in polymer electrolyte membrane (PEM) fuel cells and is the major motivation of the present work. Laboratory characterization has been conducted to determine the mechanical properties of a proton exchange membrane with respect to temperature and relative humidity. Data recovered in these tests along with properties from literature have been used in finite element models to predict the behavior of membranes used in certain applications and geometries. The overall goal of this investigation was to characterize the mechanical response of ionomer membranes in in-plane constraint configurations subjected to variable hygro-thermal environments. Expansion/contraction mechanical response of the constrained membrane as a result of change in hydration and temperature is studied in uniform and non-uniform geometries and environments. With this information, mechanical failure modes can be analyzed which is necessary for durability modeling and life prediction. The present work concentrates on defining and understanding the basic mechanical behavior of ionomeric membranes clamped in a rigid frame, and subjected to changes in temperature and humidification.

We report a systematic and quantitative structure-function relationship study of the major N-[deoxyguanosin-8-yl]-2-aminofluorene adduct (AF) derived from the prototype carcinogen 2-aminofluorene and its derivatives. The AF adduct is known to exist in two distinct conformational motifs, depending upon the location of the hydrophobic fluorine moiety: major groove binding "B type" (B) conformation (AF-dG anti ) and base-displaced "stacked" (S) conformation (AF-dG syn ). The AF-induced S/B conformational heterogeneity is sequence-dependent and follows a typical two-site dynamic chemical exchange. The population of S conformation decreases in the order of 3'-G > A » C > T, indicating the importance of the purine flanking bases in promoting the stacking structure. Line-shape analysis showed that the S/B interconversion energy barriers (ΔG ‡ ) are in the narrow 14-16 kcal/mol range. The energy differences of the two conformers are relatively small (

Human XPA is an important DNA damage recognition protein in nucleotide excision repair (NER). We previously observed that XPA binds to the DNA lesion as a homodimer [Liu, Y., Liu, Y., Yang, Z., Utzat, C., Wang, G., Basu, A. K., and Zou, Y. (2005) Biochemistry 44, 7361-7368]. Herein we report that XPA recognized undamaged DNA double-strand/single-strand (ds-ssDNA) junctions containing ssDNA branches with binding affinity (Kd = 49.1 +/- 5.1 nM) much higher than its ability to bind to DNA damage. The recognized DNA junction structures include the Y-shape junction (with both 3'- and 5'-ssDNA branches), 3'-overhang junction (with a 3'-ssDNA branch), and 5'-overhang junction (with a 5'-ssDNA branch). Using gel filtration chromatography and gel mobility shift assays, we showed that the highly efficient binding appeared to be carried out by the XPA monomer and that the binding was largely independent of RPA. Furthermore, XPA efficiently bound to six-nucleotide mismatched DNA bubble substrates with or without DNA adducts including C8 guanine adducts of AF, AAF, and AP and the T[6,4]T photoproducts. Using a set of defined DNA substrates with varying degrees of DNA bending, we also found that the XPC-HR23B complex recognized DNA bending, whereas neither XPA nor the XPA-RPA complex could bind to bent DNA. We propose that, besides DNA damage recognition, XPA may also play a novel role in stabilizing, via its high affinity to ds-ssDNA junctions, the DNA strand opening surrounding the lesion for stable formation of preincision NER intermediates. Our results provide a plausible mechanistic interpretation for the indispensable requirement of XPA for both global genome and transcription-coupled repairs. Since ds-ssDNA junctions are common intermediates in many DNA metabolic pathways, the additional potential role of XPA in cellular processes is discussed.

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli (E. coli) NER enzymes, i.e., UvrA, UvrB and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G [8-5m]mC and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: The stronger the binding to UvrA, the higher the incision rate. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA toward G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5] CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing a G[8-5m]mC or G[8-5m] T and our results revealed that these two lesions destabilized the duplex, due to an increase in free energy for duplex formation at 37°C, by approximately 5.4 kcal/mol and 3.6 kcal/mol, respectively. The destabilizations to DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.

The life of proton exchange membrane fuel cells (PEMFC) is currently lim- ited by the mechanical endurance of polymer electrolyte membranes and membrane electrode assemblies (MEAs). In this paper, the authors report recent experimental and modeling work toward understanding the mechanisms of delayed mechanical failures of polymer electrolyte membranes and MEAs under relevant PEMFC operating conditions. Mechanical breach of membranes/MEAs in the form of pinholes and tears has been fre- quently observed after long-term or accelerated testing of PEMFC cells/stacks. Cata- strophic failure of cell/stack due to rapid gas crossover shortly follows the mechanical breach. Ex situ mechanical characterizations were performed on MEAs after being sub- jected to the accelerated chemical aging and relative humidity (RH) cycling tests. The results showed significant reduction of MEA ductility manifested as drastically reduced strain-to-failure of the chemically aged and RH-cycled MEAs. Postmortem analysis re- vealed the formation and growth of mechanical defects such as cracks and crazing in the membranes and MEAs. A finite element model was used to estimate stress/strain states of an edge-constrained MEA under rapid RH variations. Damage metrics for accelerated testing and life prediction of PEMFCs are discussed. V C 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 2346-2357, 2006

A sequential Diels—Alder reaction/rearrangement of 1,3-dienes with α,β-unsaturated aldehydes is developed to form bicyclo[2.2.1]heptanone derivatives.

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.

Nucleotide excision repair (NER) plays an important role in maintaining the integrity of DNA by removing various types of bulky or distorting DNA adducts in both prokaryotic and eukaryotic cells. In Escherichia coli, the excision repair proteins UvrA, UvrB, and UvrC recognize and incise the bulky DNA damages induced by UV light and chemical carcinogens. In this process, when a putative lesion in DNA is identified initially by UvrA, a subsequent strand opening is carried out by UvrB that not only ensures that the distortion is indeed due to a damaged nucleotide but also recognizes the chemical structure of the modified nucleotides with varying efficiencies. UvrB also recruits UvrC that catalyzes both the 3'- and the 5'-incisions. Herein, we examined the interaction of UvrABC with a DNA substrate containing a single G[8,5-Me]T cross-link and compared it with T[6,4]T (the 6-4 pyrimidine-pyrimidone photoproduct) and the C8 guanine adduct of N-acetyl-2-aminofluorene (AAF). The intrastrand vicinal cross-link G[8,5-Me]T containing a covalent bond between the C8 position of guanine and the 5-methyl carbon of the 3'-thymine is formed by X-radiation, while T[6,4]T is a vicinal cross-link induced by the UV light. We also selected the AAF adduct for comparison because it represents a highly distorting monoadduct containing a covalent linkage at the C8 position of guanine. The dissociation constants (K(d)) for UvrA protein binding to DNA substrates containing the G[8,5-Me]T, T[6,4]T, and AAF adducts, as determined by gel mobility shift assays, were 3.1 +/- 1.3, 2.8 +/- 0.9, and 8.2 +/- 1.9, respectively. Although UvrA had a considerably higher affinity for G[8,5-Me]T than for the AAF adduct, the G[8,5-Me]T intrastrand cross-link was incised by UvrABC much less efficiently than the T[6,4]T intrastrand cross-link and the AAF adduct. Similar incision results also were obtained with the DNA substrates containing the adducts in a six-nucleotide bubble, indicating that the inefficient incision of G[8,5-Me]T cross-link by UvrABC was probably due to the lack of efficient recognition of the adduct by UvrB at the second step of DNA damage recognition in the E. coli NER. Indeed, as compared to T[6,4]T and AAF substrates, which clearly showed UvrB-DNA complex formation, very little UvrB complex was detectable with the G[8,5-Me]T substrate. Our result suggests that G[8,5-Me]T intrastrand cross-link is more resistant to excision repair in comparison with the T[6,4]T and AAF adducts and thus will likely persist longer in E. coli cells.

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-kDa, 32-kDa, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1 and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. In this report, we presented the evidence that Rad9-Rad1-Hus1 complex directly interacted with RPA in human cells, and this interaction was mediated by the binding of Rad9 protein to both RPA70 and RPA32 subunits. In addition, the cellular interaction of Rad9-Rad1-Hus1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose dependent manner. Such treatments also resulted in the co-localization of the nuclear foci formed with the two complexes. Consistently, knockdown of the RPA expression in cells by the small interference RNA (siRNA) blocked the DNA damage-dependent chromatin association of Rad9-Rad1-Hus1, and also inhibited the Rad9-Rad1-Hus1 complex formation. Taken together, our results suggest that Rad9-Rad1-Hus1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of Rad9-Rad1-Hus1 complex in the cellular checkpoint network.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-aminopyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA, the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8–9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial cocrystal structure. In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 DBD-AB-ssDNA complex but also revealed new protein contacts in the nucleoprotein complex. In addition to two residues, K263 and K343 of p70, previously identified by cocrystallography as direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA binding domain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259, are positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA interdomain structural reorganization induced by ssDNA binding.

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.

XPA plays an important role in the DNA damage recognition during human nucleotide excision repair. Here we report that the XPA is a homodimer either in the free state or as a complex with human RPA in solution under normal conditions. The human XPA protein purified from baculovirus-infected sf21 insect cells has a molecular mass of 36 317 Da, as determined by mass spectroscopy. However, the apparent molecular mass of XPA determined by the native gel filtration chromatography was about 71 kDa, suggesting that XPA is a dimer. This observation was supported by a native PFO-PAGE and fluorescence spectroscopy analysis. XPA formed a dimer (XPA2) in a broad range of XPA and NaCl concentrations, and the dimerization was not due to the disulfide bond formation. Furthermore, a titration analysis of the binding of XPA to the human RPA indicated that it was the XPA2 that formed the complex with RPA. Finally, the difference between the mass spectrometric and the calculated masses of XPA implies that the protein contains posttranslational modifications. Taken together, our data suggest that the dimerization of XPA may play an important role in the DNA damage recognition of nucleotide excision repair.

DNA–protein crosslinks (DPCs) arise in biological systems as a result of exposure to a variety of chemical and physical agents, many of which are known or suspected carcinogens. The biochemical pathways for the recognition and repair of these lesions are not well understood in part because of methodological difficulties in creating site-specific DPCs. Here, a strategy for obtaining site-specific DPCs is presented, and in vitro interactions of the Escherichia coli nucleotide excision repair (NER) UvrABC nuclease at sites of DPCs are investigated. To create site-specific DPCs, the catalytic chemistry of the T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase (T4-pdg) has been exploited, namely, its ability to be covalently trapped to apurinic/apyrimidinic sites within duplex DNA under reducing conditions. Incubation of the DPCs with UvrABC proteins resulted in DNA incision at the 8th phosphate 5′ and the 5th and 6th phosphates 3′ to the protein-adducted site, generating as a major product of the reaction a 12-mer DNA fragment crosslinked with the protein. The incision occurred only in the presence of all three protein subunits, and no incisions were observed in the nondamaged complementary strand. The UvrABC nuclease incises DPCs with a moderate efficiency. The proper assembly and catalytic function of the NER complex on DNA containing a covalently attached 16-kDa protein suggest that the NER pathway may be involved in DPC repair and that at least some subset of DPCs can be removed by this mechanism without prior proteolytic degradation.