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Lysosomal polarity is considered a key indicator of lysosomal function due to its significant impact on membrane fluidity and enzymatic reactions in lysosomes. Monitoring lysosomal polarity can gain insight into the related physiological and pathological processes and develop new diagnostic methods. However, current fluorescent probes with lysosomal polarity response suffer from narrow linear range, photobleaching and complicated preparation. Herein, a ratiometric fluorescent probe (r-bCDs) for intracellular lysosomal polarity imaging is designed and constructed by amide bond assembly of polarity-sensitive red fluorescent carbon dots (rCDs) and referenced blue fluorescent carbon dots (bCDs). r-bCDs show a much wider linear range of polarity response (orientation polarizability Δf from 0.020 to 0.315) than other probes, and the interference of uneven distribution and instrument factors can be effectively eliminated by ratiometric fluorescent sensing. Imaging of intracellular lysosomal polarity with r-bCDs is implemented to observe the polarity variation caused by the change of cell state and the difference between cancer cells and normal cells. This work provides a promising tool for studying the related physiological and pathological processes and developing new diagnostic methods.

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

Condensable particulate matter (CPM) is a major component of primary particulate matter emitted into the atmosphere from stationary sources. However, the factors affecting CPM generation remain unclear. In this study, we systematically investigated the role of sulphur and chlorine in CPM formation during coal combustion. To explore the influence of S, various concentrations of SO

With the aim of discovering novel nematicidal scaffolds, the nematicidal activities of a series of quinazoline compounds were tested, with some compounds showing excellent results. Among them, the LC

Dictyostelid cellular slime molds are a ubiquitous component of most soils, where they feed upon bacteria and other microbes and thus play an essential role in the soil ecosystem. Herein we review the available literature on dictyostelid cellular slime molds in China, especially their diversity and ecology. The patterns of distribution for these organisms in relation to the different habitats with which they are associated are analyzed and discussed. In addition, the assemblages of dictyostelids reported from China and the United States were compared. The CC value obtained (0.48) indicates that China and the United States are perhaps less similar than might be expected. Our data point out the need for further studies to characterize more completely the assemblages of dictyostelids associated with particular vegetation types or particular regions throughout the world.

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.

Anaerobic digestates were potential mediums for cultivating oleaginous microalgae, but their various components brought uncertainties for aglal growth and lipids production. In this study, three microalgae strains were tested to grow on four typical anaerobic digestates. The results showed that anaerobic food wastewater was an optimal medium for C. pyrenoidosa and S. obliquus culture (N. oleoabundanst cannot survive), achieving the highest biomass (2.15-2.32 g L

In this study, ultrasonic effects on structure, chain conformation and morphology of pectin extracted from Premna microphylla Turcz (PEP) and its probable mechanism were investigated. In the process of ultrasonic treatments, the chains of PEP were fractured rapidly within the initial 10 min and then the degradation rate gradually slowed down. The primary structure of PEP nearly remained unchanged after ultrasonic degradation. The rigid semi-flexible chains of PEP were converted into flexible chains, flexible coils, even compact coils. Sonication at low intensity for short time made PEP molecular chains curly collapse and tighten up. Long duration sonication at high intensity generated excessive small rigidness segments that mutually aggregated because of hydrogen bonds and inhibited the self-coiling of PEP chains. Atomic force microscopy (AFM) analysis supported the conformation transition of PEP chains. The results provided a fundamental basis for orientation design and process control of PEP structure.

Expression of the immune checkpoint programmed death ligand-1 (PD-L1) is increased in ovarian cancer (OC) and correlates with poor prognosis. Interferon-γ (IFNγ) induces PD-L1 expression in OC cells, resulting in their increased proliferation and tumor growth, but the mechanisms that regulate the PD-L1 expression in OC remain unclear. Here, we show that the IFNγ-induced PD-L1 expression in OC cells is associated with increased levels of STAT1, Tyr-701 pSTAT1 and Ser-727 pSTAT1. Suppression of JAK1 and STAT1 significantly decreases the IFNγ-induced PD-L1 expression in OC cells, and STAT1 overexpression increases the IFNγ-induced PD-L1 expression. In addition, IFNγ induces expression of the transcription factor interferon regulatory factor 1 (IRF1) and IRF1 suppression attenuates the IFNγ-induced gene and protein levels of PD-L1. Chromatin immunoprecipitation results show that IFNγ induces PD-L1 promoter acetylation and recruitment of STAT1, Ser-727 pSTAT1 and IRF1 in OC cells. Together, these findings demonstrate that the IFNγ-induced PD-L1 expression in OC cells is regulated by JAK1, STAT1, and IRF1 signaling, and suggest that targeting the JAK1/ STAT1/IRF1 pathway may provide a leverage to regulate the PD-L1 levels in ovarian cancer.

In this study, the heat product (90 °C, 10 min) of β-lactoglobulin (β-LG) was analyzed by asymmetric-flow field-flow fractionation (AF4) to observe the effect of heat treatment. The changes in molar mass (M) and molar size induced by heat treatment were characterized by AF4, and changes in molar shape were observed by transmission electron microscopy (TEM). The results showed that β-LG dissociated and aggregated into four fractions with different M values, sizes, and shapes after heat treatment. The vast aggregations with the highest allergenicity (IgE-binding capacity) might enhance the allergenicity of β-LG. However, the number of characterized epitope peptides was decreased due to heat treatment. The above results provide some references for related studies of β-LG and its allergenicity. Further separation and characterization of the high-allergenicity fractions and peptides will help to eliminate allergens in dairy products and reduce the occurrence of allergic reactions.

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.

Interferon-γ (IFNγ) is a pleiotropic cytokine that has a crucial role in immune response and tumor immunity. Because of its anti-tumor effects, IFNγ has been used in cancer treatment. However, IFNγ also has tumor-promoting functions that are less well understood. Here, we show that IFNγ induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) in ovarian cancer (OC) cells. The IFNγ-induced IL-8 expression is dependent on JAK1, STAT1, and p65 NFκB, and is associated with an increased occupancy of K314/315 acetylated p65 NFκB and Ser-727 phosphorylated STAT1 at the IL-8 promoter. Neutralization of IL-8 using anti-IL-8 antibody reduces IFNγ-induced migration of OC cells, and their invasion ability in 3D spheroids. Together, these findings identify IL-8 as a novel target induced by IFNγ/JAK1/STAT1/p65 NFκB signaling, and indicate that the IFNγ-induced IL-8 contributes to IFNγ pro-tumorigenic effects in ovarian cancer cells.

Reflectance Anisotropy (RA) contains crucial information about the optical behavior and structure of ground objects and is typically described by the Bidirectional Reflectance Distribution Function (BRDF). However, traditional RA-retrieval methods from satellites or the ground are constrained due to the low spatial resolution, illumination conditions, and instrument-associated limitations. In this study, Unmanned Aerial Vehicle (UAV) based oblique photography technology is applied to RA retrieval, aiming to explore the feasibility of UAV to obtain spatially continuous RA data with high spatial resolution and high accuracy. The Monte Carlo method is used to select and optimize the combination of multi-angle observation data of a variety of ground objects obtained by UAV-based oblique photogrammetry. The accuracy and applicability of two BRDF inversion models, namely the linear semi-empirical kernel-driven (LSEKD) model and the non-linear Rahman-Pinty-Verstraete (RPV) model, are analyzed, and the effects of different sampling window sizes on the BRDF results of various ground objects are compared. The main conclusions are: 1) Oblique photogrammetry is a highly efficient method for RA measurement at the centimeter-level resolution for spatially continuous regions, because it enables cameras with a narrow field of view (FOV) to obtain observation data with a wider viewing angle and more directions, ensuring the robustness and accuracy of the BRDF inversion model, with RMSE in the visible and near-infrared bands are about 0.003 and 0.019 (8–14%); 2) Both the LSEKD and the RPV model are suitable for the inversion of the BRDF with comparable accuracy, but the kernel functions for the LSEKD need to be carefully chosen in advance according to the characteristics of the ground object, while the RPV is adaptive for most objects; 3) The Monte Carlo method can ensure that the multi-angle observation data is distributed evenly in the hemisphere, thus providing the optimal dataset for a high-accuracy BRDF inversion; and 4) The RA of ground objects may change with the change of spatial resolution; the extremely high-resolution RA provides the ability to study ground objects at a finer scale. This study expands the methods for obtaining high-spatial resolution, high-accuracy, and spatially continuous RA. It has significant application potential in areas such as quantitative remote sensing, calibration of high-accurate remote-sensing products, and bridging the scale gap between satellite and ground-collected RA data.

The return level is an important measure for the extreme precipitation, and maps of the return level are used to derive information for the design of hydrological and hydraulic engineering projects. The interpolation of hourly extreme precipitation is challenging due to low station density and high inhomogeneity. Hourly precipitation observations at 232 weather stations in the Haihe River basin from 1961 to 2012 were used to evaluate six interpolation methods including Inverse Distance Weighted (IDW), Ordinary Kriging (OK), Kriging with External Drift (KED) assisted by different covariables and Empirical Bayesian Kriging (EBK) for generating 2-, 5-, 10-, 20-, 50-, and 100-year return level maps for the basin. Leave-one-out cross-validation showed that although there was a high correlation coefficient between hourly extreme precipitation and the elevation (DEM), KED incorporating DEM as the covariable (KED_DEM) did not improve the interpolation efficiency compared with OK. KED with the annual average precipitation as the covariable (KED_AP), which had the largest Nash-Sutcliffe efficiency coefficient (NSE, 0.89–0.45) and the smallest root mean square error (RMSE, 2.23–15.05 mm) for the six return levels, outperformed the other five methods compared. The return levels varied from 13 to 46, 20–66, 24–78, 26–89, 29–110 and 30–134 mm, with mean values of 30, 40, 48, 57, 67 and 75 mm for the 2-, 5-, 10-, 20-, 50- and 100-year return levels, respectively, in the Haihe River basin. The return level maps of extreme hourly precipitation in the Haihe River basin were generated based on the KED_AP method and showed a spatial distribution of decreasing trend from the southeastern part to the northwestern part of the basin. The high-value centre moved from the eastern coastal area for smaller return level maps to the southern area for the 50- and 100-year return level maps. This study may provide some insights into the spatial interpolation of extreme precipitation on an hourly scale.

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.

Quantifying the influence of human activities on hydrological drought is of great significance to sustainable water resource management, but it remains difficult. The key to solving this problem is determining how to scientifically select the natural reference tributary. On the basis of existing research, this paper puts forward a tributary-comparison method that combines the Drought Propagation Intensity Index (DPI) with the human activity index to preliminarily and quantitatively determine the natural reference tributary from among all tributaries and then quantify the influence of human activities on hydrological drought in other tributaries. The empirical research was carried out in the upper reaches of the Shiyang River in the eastern area of Qilian Mountain. The results showed that, firstly, at the annual and seasonal scales, the tributaries in the upper reaches of the Shiyang River changed from hydrological humidification to hydrological aridification, from west to east, from 1961 to 2016. On the whole, the hydrological aridification of Huangyang River in the central part was the most serious, but its hydrological drought duration and drought Intensity showed a downward trend at a monthly scale. Secondly, using the tributary-comparison method, Xiying River was quantitatively selected as the natural reference tributary, and the influence of human activities on hydrological drought in other tributaries was quantified. On the whole, human activities will reduce hydrological drought duration and enhance hydrological drought intensity. The empirical study further proved that the tributary-comparison method has strong applicability in multi-tributary areas with obvious differences in human activities and provides a useful supplement to existing research methods. According to this method, tributaries that are weakly affected by human activity (or not affected at all) can be selected, and the tributaries least affected by human activity can also be identified. On this basis, the influence degree and direction of human activities in other tributaries on hydrological drought can be quantified.

The silver-deposited silicon (Si/Ag) is a successful strategy to enhance the conductivity of Si anode material. However, Ag nanoparticles can be separated from the Si due to the huge volume variation, which causes the deterioration of cycling performance. In this research, we propose a facile method of synthesizing Si/Ag particles with alloyed interface (Si/Ag2Si/Ag) by rapid thermal process to enhance the mechanical contact between Si and Ag. The reversible capacity of Si/Ag2Si/Ag still remains 1138 mAh g−1 over 100 cycles at 0.5 C (1 C = 4.2 A g−1), which is 267 mAh g−1 higher than of Si/Ag. Besides, Si/Ag2Si/Ag holds the reversible capacity of 849 mAh g−1 at 2 C.

Aiming at solving differential equations of plane problems, the algorithm of difference equation is established, and the corresponding program is compiled on BP neural network. The correctness and practicability of the difference equation algorithm are verified. A dynamic model of the parallel difference equation is constructed according to the characteristics of the parallel structure of BP neural network. By calculating examples, the continuity condition under the condition of modulus abruption is further discussed. The study shows that the two groups of differential equations are used to identify and verify the model, and the energy function satisfies both the linear embedding condition and the correct wiring. Furthermore, BP neural network is used to realize the search and routing of the maximum plane. The results show that difference equation calculations have the ability to help BP networks get rid of local minima and get better results.

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.

Although there have been many studies on atomic layer deposition of Al2O3 to improve the electrochemical performances of ternary cathode materials, few studies considered the harsh conditions such as high temperature and high voltage at the same time. In this work, the Al2O3 passivation layer is employed to coat the high nickel (Ni) LiNi0.68Co0.10Mn0.22O2 cathode through atomic layer deposition (ALD) approach. The Al2O3 coated LiNi0.68Co0.10Mn0.22O2 presents superior cycling stability and rate capability than the pristine at the cut-off voltage of 2.7–4.4 V under room temperature. Furthermore, under the harsh condition at high temperature of 60 °C or at high voltage of 2.7–4.8 V, the Al2O3 coated LiNi0.68Co0.10Mn0.22O2 also shows significantly improved performances than the pristine. The superior electrochemical performance of Al2O3 coated LiNi0.68Co0.10Mn0.22O2 could be attributed to the enhanced interfacial stability, the mitigated side reactions with electrolyte and the better maintained lithium ion diffusion kinetics. Manipulating the surface coating chemistry and evaluating the performance under harsh condition provide a useful approach for fast screening of effective surface modification technology, which might significantly advance the development of high energy density cathode materials.

Oxygen vacancy (VO) is one of the most important defects and acts as the reactive sites in a variety of catalytic reactions. In this work, doping method is employed to generate controllable VO in ZnWO4 crystal and the existence of VO was validated by TEM, XPS and UV–vis absorbance spectra. A novel electrochemical sensor based on Ce-doped ZnWO4 modified carbon paste electrode (CPE) has been fabricated and the electrochemical performance of the proposed sensor is verified by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The as-prepared oxygen vacancy-rich ZnWO4 shows excellent electrocatalytic performance in sensing carbendazim (CBZ) compared to the virgin CPE. The results demonstrate that VO can effectively enhance electronic conductivity and narrow the band gap, leading to superior performances of 2%Ce-doped ZnWO4 in electrocatalytic reactions. Low detection limits (LOD) of CBZ is obtained to be 0.0033 μmol L−1 via DPV method (S/N = 3). The impressive detection performance is even spread to the simultaneous determination of common neurotransmitters, DA an UA, with detection limits of 0.0033 μmol L−1 and 0.17 μmol L−1 and wide linear range from 0.01 to 30 μmol L−1 and 0.5 to 100 μmol L−1 for DA and UA, respectively.

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 melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.

ATR, a PI3K-like protein kinase, plays a key role in regulating DNA damage responses. Its nuclear checkpoint kinase function is well documented, but little is known about its function outside the nucleus. Here we report that ATR has an antiapoptotic activity at mitochondria in response to UV damage, and this activity is independent of its hallmark checkpoint/kinase activity and partner ATRIP. ATR contains a BH3-like domain that allows ATR-tBid interaction at mitochondria, suppressing cytochrome c release and apoptosis. This mitochondrial activity of ATR is downregulated by Pin1 that isomerizes ATR from cis-isomer to trans-isomer at the phosphorylated Ser428-Pro429 motif. However, UV inactivates Pin1 via DAPK1, stabilizing the pro-survival cis-isomeric ATR. In contrast, nuclear ATR remains in the trans-isoform disregarding UV. This cytoplasmic response of ATR may provide a mechanism for the observed antiapoptotic role of ATR in suppressing carcinogenesis and its inhibition in sensitizing anticancer agents for killing of cancer cells.

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.

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.

Human replication protein A (RPA), composed of RPA70, RPA32, and RPA14 subunits, undergoes hyperphosphorylation in cells in response to DNA damage. Hyperphosphorylation that occurs predominately in the N-terminal region of RPA32 is believed to play a role in modulating the cellular activities of RPA essential for almost all DNA metabolic pathways. To understand how the hyperphosphorylation modulates the functions of RPA, we compared the structural characteristics of full-length native and hyperphosphorylated RPAs using mass spectrometric protein footprinting, fluorescence spectroscopy, and limited proteolysis. Our mass spectrometric data showed that of 24 lysines and 18 arginines readily susceptible to small chemical reagent modification in native RPA, the three residues Lys-343, Arg-335, and Arg-382, located in DNA binding domain B (DBD-B) of RPA70, were significantly shielded in the hyperphosphorylated protein. Tryptophan fluorescence studies indicated significant quenching of Trp-361, located in the DBD-B domain, induced by hyperphosphorylation of RPA. Consistently, DBD-B became more resistant to the limited proteolysis by chymotrypsin after RPA hyperphosphorylation. Taken together, our results indicate that upon hyperphosphorylation of RPA32 N terminus (RPA32N), RPA undergoes a conformational change involving the single-stranded DNA binding cleft of DBD-B. Comparison of the interactions of native and hyperphosphorylated RPAs with short single-stranded oligonucleotides or partial DNA duplexes with a short 5' or 3' single-stranded DNA tails showed reduced affinity for the latter protein. We propose that the hyperphosphorylation may play a role in modulating the cellular path-ways by altering the DBD-B-mediated RPA-DNA and RPA-protein interactions, hypothetically via the interaction of hyperphosphorylated RPA32N with DBD-B.

To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon. The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli. Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic. Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E. coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links. The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E. coli UvrABC complex. UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system.

UvrA is one of the key Escherichia coli proteins involved in removing DNA damage during the process of nucleotide excision repair. The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions. In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E. colimolecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE. These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision. Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity. Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP. UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type strain. Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6–4 photoproduct removal in the DnaK-deficient cells. These results suggest that molecular chaperonins participate in nucleotide excision repair by maintaining repair proteins in their properly folded state.

The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed. DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.

Dinuclear bis(platinum) cations, containing as potential leaving group dimethylsulfoxide (Me2SO), were prepared. The general formula is [{trans-Pt(Me2SO)(NH3)2}2NH2(CH2)nNH2]4+. These complexes were found to induce the Z form of poly(dG-dC).poly(dG-dC). The complexes also appear to bind to DNA forming interstrand cross-links by displacement of Me2SO. In aqueous solution, rapid hydrolysis occurs to free diamine and the trans-[Pt(H2O)(Me2SO)(NH3)2]2+ cation. In a surprising finding, this monomeric species (as the chloride trans-[PtCl(Me2SO)(NH3)2]+) was found to be a much more efficient interstrand cross-linking agent than the parent trans-[PtCl2(NH3)2]. Thus, steric control by bulky ligands such as Me2SO may be used to affect relative tendencies to inter- or intrastrand cross-link formation in platinum complexes.

The carbonyl cofactor of bovine plasma amine oxidase (EC 1.4.3.6), recently shown to be 6-hydroxydopa (also known as topa), has been spin labeled to the extent of one label per enzyme dimer molecule, using 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) and 4-hydrazino-TEMPO followed by reduction with borohydride. By studying the EPR spectra of the labeled enzyme, it has been deduced that there is no magnetic interaction between the copper and the spin label, and that the spin label is at least 1.3 nm distant from the copper(II) ion in the resting enzyme. The bound label is strongly immobilized, is in a sterically constricted environment, and is not accessible to small anions. Removal of the copper does not alter the EPR spectrum of the label. The results are similar to results for porcine plasma amine oxidase, and show that the copper is not close to, and does not directly interact with, the topa-bound substrate.