The male reproductive toxicity after 5-Fluorouracil exposure: DNA damage, oxidative stress, and mitochondrial dysfunction in vitro and in vivo.

5-Fluorouracil (5-FU), a chemotherapeutic drug used to treat a variety of cancers, can enter the environment through different routes, causing serious public health and environmental concerns. It has been reported that 5-FU exposure adversely affects male reproductive function, and its effects on this system cannot be avoided. In this study, using western blotting and quantitative polymerase chain reaction studies, we found that 5-FU promoted testicular injury by inducing oxidative stress, which was accompanied by the inhibition of nuclear factor erythroid 2-related factor 2/antioxidant response element signaling. Accumulation of reactive oxygen species (ROS) aggravated 5-FU-mediated mitochondrial dysfunction and apoptosis in murine cell lines and testes, indicating oxidative stress and mitochondrial-dependent apoptotic signaling play crucial roles in the damage of spermatogenic cells caused. N-Acetyl-L-cysteine, an antioxidant that scavenges intracellular ROS, protected spermatogenic cells from 5-FU-induced oxidative damage and mitochondrial dysfunction, revealing the important role of ROS in testicular dysfunction caused by 5-FU. We found that 5-FU exposure induces testicular cell apoptosis through ROS-mediated mitochondria pathway in mice. In summary, our findings revealed the reproductive toxicological effect of 5-FU on mice and its mechanism, provided basic data reference for adverse ecological and human health outcomes associated with 5-FU contamination or poisoning.

pparß regulates lipid catabolism by mediating acox and cpt-1 genes in Scophthalmus maximus under heat stress.

Peroxisome proliferator-activated receptor ß (pparß) is a key gene-regulating lipid metabolism pathway, but its function in turbot remains unclear. In this study, the CDS of pparß was cloned from kidney for the first time. The CDS sequence length was 1533 bp encoding 510 amino acids. Structural analysis showed that the pparß protein contained a C4 zinc finger and HOLI domain, suggesting that the pparß gene of turbot has high homology with the PPAR gene of other species. The high expression patterns of pparß, acox, and cpt-1 at high temperatures, as shown through qPCR, indicated that high temperatures activated the transcriptional activity of pparß and increased the activity of the acox and cpt-1 genes. The expression of acox and cpt-1 was significantly inhibited when pparß was downregulated using RNAi technology and inhibitor treatments, suggesting that pparß positively regulated acox and cpt-1 expression at high temperatures and, thus, modulates lipid catabolism activity. These results demonstrate that pparß is involved in the regulation of lipid metabolism at high temperatures and expand a new perspective for studying the regulation of lipid metabolism in stress environments of teleost.

Three-Dimensional Quantitative Coherent Diffraction Imaging of Staphylococcus aureus Treated with Peptide-Mineralized Au-Cluster Probes.

Characterizing interactions between microbial cells and their specific inhibitory drugs is essential for developing effective drugs and understanding the therapeutic mechanism. Functional metal nanoclusters can be effective inhibitory agents against microorganisms according to various characterization methods, but quantitative three-dimensional (3D) spatial structural analysis of intact cells is lacking. Herein, using coherent X-ray diffraction imaging, we performed in situ 3D visualization of unstained Staphylococcus aureus cells treated with peptide-mineralized Au-cluster probes at a resolution of ∼47 nm. Subsequent 3D mass-density mapping and quantitative structural analyses of S. aureus in different degrees of destruction showed that the bacterial cell wall was damaged and cytoplasmic constituents were released from cells, confirming the significant antibacterial effects of the Au-cluster probe. This study provides a promising nondestructive approach for quantitative imaging and paves the way for further research into microbe-inhibitor drug interactions.

Structural and functional characterization of turbot pparγ: Activation during high temperature and regulation of lipid metabolism.

Transcription factors of the peroxisome proliferator-activated receptor gamma (pparγ) is a ligand-activated receptor that plays key roles in lipid metabolism and inflammation. In this study, we cloned the pparγ cDNA of turbot (Scophthalmus maximus). It has a 1659 bp coding sequence (CDS) and encodes 552 amino acids. The deduced PPARγ protein of turbot contains two highly conserved domains, a C4-type zinc finger, a nuclear hormone receptor DNA-binding region signature, and a HOLI domain (ligand-binding domain of hormone receptors) identical to that of in other species. qPCR results showed that the expression level of pparγ and the expression of the fatty acid transporters fatp and cd36 were significantly increased under high-temperature stress. This indicates that high temperatures activate the transcriptional activity of pparγ, and lipid metabolism plays an important role in turbot under high-temperature stress. In addition, RNA interference was used to explore the regulation of pparγ on lipid metabolism of turbot at high temperatures. The results showed that the mRNA level of pparγ was significantly decreased. After the expression level of pparγ was inhibited, the expression levels of fatp and cd36 were significantly decreased. At the same time, GW9662 (a pparγ antagonist) was used to inhibit pparγ activity in turbot kidney cells, and fatp and cd36 gene expressions were detected. The mRNA expression levels of pparγ, fatp, and cd36 were significantly decreased. Our results suggest that high temperatures activate pparγ in turbot and that pparγ regulates lipid transport and maintains lipid metabolism homeostasis through positive regulation of the expression of downstream genes fatp and cd36. This study further elucidates that the pparγ-mediated signaling pathway may play an important role in regulating lipid metabolism during heat stress in teleost fish.

Dissecting the Molecular Regulation of Natural Variation in Growth and Senescence of Two Eutrema salsugineum Ecotypes.

Salt cress (Eutrema salsugineum, aka Thellungiella salsuginea) is an extremophile and a close relative of Arabidopsis thaliana. To understand the mechanism of selection of complex traits under natural variation, we analyzed the physiological and proteomic differences between Shandong (SD) and Xinjiang (XJ) ecotypes. The SD ecotype has dark green leaves, short and flat leaves, and more conspicuous taproots, and the XJ ecotype had greater biomass and showed clear signs of senescence or leaf shedding with age. After 2-DE separation and ESI-MS/MS identification, between 25 and 28 differentially expressed protein spots were identified in shoots and roots, respectively. The proteins identified in shoots are mainly involved in cellular metabolic processes, stress responses, responses to abiotic stimuli, and aging responses, while those identified in roots are mainly involved in small-molecule metabolic processes, oxidation-reduction processes, and responses to abiotic stimuli. Our data revealed the evolutionary differences at the protein level between these two ecotypes. Namely, in the evolution of salt tolerance, the SD ecotype highly expressed some stress-related proteins to structurally adapt to the high salt environment in the Yellow River Delta, whereas the XJ ecotype utilizes the specialized energy metabolism to support this evolution of the short-lived xerophytes in the Xinjiang region.

Ultrafast Single-Particle Imaging with Intense X-Ray Pulses.

Ultrafast single-particle imaging with intense x-ray pulses from free-electron laser sources provides a new approach for visualizing structure and dynamics on the nanoscale. After a short introduction to the novel free-electron laser sources and methods, we highlight selected applications and discuss how ultrafast imaging flourishes from method development to early applications in physics and biology to opportunities for chemical sciences.

Genetic parameters of seven immune factors in turbot (Scophthalmus maximus) infected with Vibrio anguillarum.

In this study, 1,800 turbot (Scophthalmus maximus) individuals from 30 full-sib families were experimentally infected with Vibrio anguillarum, and the expression levels of the immune factors lysozyme, hepcidin, heat-shock protein 70 (HSP70), HSP90, immunoglobulin M (IgM), C-type lectin and Lily-type lectin in the liver were measured by real-time PCR. Heritability values of the seven immune factors were 0.289 ± 0.087, 0.092 ± 0.024, 0.282 ± 0.043, 0.244 ± 0.027, 0.343 ± 0.081, 0.092 ± 0.011 and 0.084 ± 0.009, respectively. The ranges of phenotypic, genetic and environmental correlations were -0.889 to 0.759, -0.841 to 0.888 and -0.919 to 0.883, respectively. The heritability values of HSP70, HSP90 and IgM were moderate, and the genetic correlations between HSP70, HSP90 and IgM were moderate to highly positive, which suggests that the immunocompetence of turbot against V. anguillarum can be improved by genetically improving these three immune characters via multi-trait integrated breeding technology or indirect selection.

Edge re-projection method for high-quality edge reconstruction in non-line-of-sight imaging.

How to image scenes or detect objects hidden from view has been of increasing interest in recent years. Previous studies have demonstrated non-line-of-sight object reconstruction by using time-resolved detectors and a back-projection algorithm, whereas the filtered back-projection method reconstructs high-frequency spatial information, such as the edge of an object, with poor quality. Here we propose an optimized back-projection algorithm to improve the object edge reconstruction quality. We base our method on the observation that the spatial frequency and geometric information required to reconstruct an edge is distributed unevenly across scanning positions of the relay wall. Our method extracts edge voxels from the first projection result, correcting the signal response weight at different scanning positions according to their relative contributions to the object edge reconstruction, and then re-projects data. Simulations and experiments show that compared to the filtered back-projection algorithm, our method achieves better reconstruction results for the object edge, which makes it easier to distinguish the object shape.

The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source.

The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

Expression and localization study of pIgR in the late stage of embryo development in turbot (Scophthalmus maximus).

The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot.

Enhancement of phase retrieval capability in ptychography by using strongly scattering property of the probe-generating device.

In common ptychographic coherent diffractive imaging (PCDI) systems, the probe-generating devices typically exhibit strong scattering, which is not fully used. Here, we report the reasonableness of using the diffraction pattern of the probe-generating device as the frequency-domain information of the scanning probe located in the sample plane, and we propose a method introducing this frequency-domain information into an iterative process to improve the imaging quality of PCDI. The new method was demonstrated using both a visible laser source and a synchrotron radiation X-ray source; the proposed method significantly improved the imaging quality in both demonstrations.

A High-Speed Imaging Method Based on Compressive Sensing for Sound Extraction Using a Low-Speed Camera.

This paper reports an efficient method for sound extraction from high-speed light spot videos reconstructed from the coded light spot images captured with a low-speed camera based on compressive sensing, but at the expense of consuming time. The proposed method first gets the high-speed video of the light spot that is illuminated on the vibrating target caused by sound. Then the centroid of the light spot is used to recover the sound. Simulations of the proposed method are carried out and experimental results are demonstrated. The results show that high-speed videos with a frame rate of 2000 Hz can be reconstructed with a low-speed (100 Hz) charge-coupled device (CCD) camera, which is randomly modulated by a digital micro-mirror device (DMD) 20 times during each exposure time. This means a speed improvement of 20 times is achieved. The effects of synchronization between CCD image recording and DMD modulation, the optimal sampling patterns of DMD, and sound vibration amplitudes on the performance of the proposed method are evaluated. Using this compressive camera, speech (counting from one to four in Chinese) was recovered well. This has been confirmed by directly listening to the recovered sound, and the intelligibility value (0⁻1) that evaluated the similarity between them was 0.8185. Although we use this compressive camera for sound detection, we expect it to be useful in applications related to vibration and motion.

Effects of forest regeneration practices on the flux of soil CO2 after clear-cutting in subtropical China.

Reforestation after clear-cutting is used to facilitate rapid establishment of new stands. However, reforestation may cause additional soil disturbance by affecting soil temperature and moisture, thus potentially influencing soil respiration. Our aim was to compare the effects of different reforestation methods on soil CO2 flux after clear-cutting in a Chinese fir plantation in subtropical China: uncut (UC), clear-cut followed by coppicing regeneration without soil preparation (CC), clear-cut followed by coppicing regeneration and reforestation with soil preparation, tending in pits and replanting (CCRP), and clear-cut followed by coppicing regeneration and reforestation with overall soil preparation, tending and replanting (CCRO). Clear-cutting significantly increased the mean soil temperature and decreased the mean soil moisture. Compared to UC, CO2 fluxes were 19.19, 37.49 and 55.93 mg m-2 h-1 higher in CC, CCRP and CCRO, respectively (P < 0.05). Differences in CO2 fluxes were mainly attributed to changes in soil temperature, litter mass and the mixing of organic matter with mineral soil. The results suggest that, when compared to coppicing regeneration, reforestation practices result in additional CO2 released, and that regarding the CO2 emissions, soil preparation and tending in pits is a better choice than overall soil preparation and tending.

Synchrotron X-ray Microtomography with Improved Image Quality by Ring Artifacts Correction for Structural Analysis of Insects.

Ring artifacts are undesirable and complicate the analysis and interpretation of microstructures in synchrotron X-ray microtomography. Here, we propose a new method to improve the image quality of an object by removing the ring artifacts and investigate the efficiency of this process with tomographic images of a dried Tenebrio molitor. In this method, before the tomographic reconstruction, ring artifacts were identified and located in the sinograms as line artifacts. Then, the identified line artifacts were corrected as single point noise via image processing of the original projections. Eventually, the corresponding line artifacts were removed, resulting in reduced ring artifacts in the reconstructed tomographic images. Simulations verified the efficiency of the proposed method. This method was successfully applied for the structural analysis of the insect T. molitor, showing superior performance in reducing ring artifacts in the tomographic image without noticeable loss of structural information.

Immunological characterization and expression of lily-type lectin in response to environmental stress in turbot (Scophthalmus maximus).

Lectins are a superfamily of carbohydrate-binding proteins that are widely distributed throughout living organisms. In earlier work, we identified lily-type lectin (SmLTL) in the skin mucus of turbot Scophthalmus maximus, and we characterized the protein in the present study. Results from qRT-PCR indicated that SmLTL was expressed highly in skin, intestine and gill tissue. Changes in SmLTL expression occurred in these tissues in response to environmental stressors including ciliate infection, high temperature and salinity. Recombinant SmLTL purified from Escherichia coli was able to haemagglutinate mouse erythrocytes in the absence of calcium, and was inhibited by d-mannose. In addition, SmLTL displayed selective binding to bacterial species including Edwardsiella tarda and Vibrio anguillarum, and exhibited toxicity towards Philasterides dicentrarchi, with a mortality of over 60% after 24 h at a concentration of only 100 µgml-1. To investigate this toxicity further, we measured binding of SmLTL after incubating the ciliate in FITC-SmLTL solution. Surface fluorescence decreased substantially in the presence of 400 mM d-mannose. Together these results suggest that lily-type lectins serve as the first line of defence against microbial attack and play a pivotal role in the mucosal immune system.

Spatiotemporal exposure modeling of ambient erythemal ultraviolet radiation.

BACKGROUND: Ultraviolet B (UV-B) radiation plays a multifaceted role in human health, inducing DNA damage and representing the primary source of vitamin D for most humans; however, current U.S. UV exposure models are limited in spatial, temporal, and/or spectral resolution. Area-to-point (ATP) residual kriging is a geostatistical method that can be used to create a spatiotemporal exposure model by downscaling from an area- to point-level spatial resolution using fine-scale ancillary data. METHODS: A stratified ATP residual kriging approach was used to predict average July noon-time erythemal UV (UVEry) (mW/m2) biennially from 1998 to 2012 by downscaling National Aeronautics and Space Administration (NASA) Total Ozone Mapping Spectrometer (TOMS) and Ozone Monitoring Instrument (OMI) gridded remote sensing images to a 1 km spatial resolution. Ancillary data were incorporated in random intercept linear mixed-effects regression models. Modeling was performed separately within nine U.S. regions to satisfy stationarity and account for locally varying associations between UVEry and predictors. Cross-validation was used to compare ATP residual kriging models and NASA grids to UV-B Monitoring and Research Program (UVMRP) measurements (gold standard). RESULTS: Predictors included in the final regional models included surface albedo, aerosol optical depth (AOD), cloud cover, dew point, elevation, latitude, ozone, surface incoming shortwave flux, sulfur dioxide (SO2), year, and interactions between year and surface albedo, AOD, cloud cover, dew point, elevation, latitude, and SO2. ATP residual kriging models more accurately estimated UVEry at UVMRP monitoring stations on average compared to NASA grids across the contiguous U.S. (average mean absolute error [MAE] for ATP, NASA: 15.8, 20.3; average root mean square error [RMSE]: 21.3, 25.5). ATP residual kriging was associated with positive percent relative improvements in MAE (0.6-31.5%) and RMSE (3.6-29.4%) across all regions compared to NASA grids. CONCLUSIONS: ATP residual kriging incorporating fine-scale spatial predictors can provide more accurate, high-resolution UVEry estimates compared to using NASA grids and can be used in epidemiologic studies examining the health effects of ambient UV.

A novel L-asparaginase from Aquabacterium sp. A7-Y with self-cleavage activation.

We have identified a novel L-asparaginase, abASNase3, from Aquabacterium sp. A7-Y. abASNase3 is composed of 306 amino acids and exhibits 34 % sequence homology to human asparaginase (hASNase3). Further analysis revealed that abASNase3 belongs to the N-terminal nucleophile (Ntn) family of hydrolases. Previous reports about the Ntn hydrolase family and the results of our study suggest that abASNase3 must form two subunits by self-cleavage between Gly189 and Thr190 to attain catalytic activity. The two subunits remained tightly associated to build a single functional unit. The optimum pH for abASNase3 was found to be 8.0 in Tris-HCl buffer and the enzyme was found to be stable over a broad pH range from pH 6.0 to 12.0. The optimum temperature for abASNase3 was found to be approximately 40 °C, and the enzyme was stable below 65 °C. abASNase3 showed high substrate specificity toward L-asparagine and had no or only slight activity toward D-asparagine, L-glutamine and D-glutamine. abASNase3 was significantly activated by Mg(2+) and was substantially inhibited by Ni(2+), Cu(2+), Mn(2+) and Co(2+). The Michaelis-Menten constant and turnover number of abASNase3 for L-asparagine were estimated to be 3.37 × 10(-2) M and 8.72 × 10(-3) s(-1), respectively. Our results indicate that abASNase3 is a novel L-asparaginase in the Ntn family of hydrolases.

Cadmium accumulation and tolerance of Lagerstroemia indica and Lagerstroemia fauriei (Lythraceae) seedlings for phytoremediation applications.

Contamination by heavy metals is one of the most serious environmental problems generated from human activities. Because phytoremediation utilizes plants to uptake contaminants, it could potentially be used to remediate metal-contaminated areas. A pot culture experiment with four levels of cadmium (Cd) (0, 20, 40, and 80 mg of Cd/kg dry soil) was conducted to investigate Cd accumulation and tolerance of roots, shoots, and leaves of Lagerstroemia indica and Lagerstroemia fauriei as well as their potential for phytoremediation. Experimental results indicated that Cd inhibited seedling growth only at the higher Cd exposure concentration (40 and 80 mg/kg). The tolerance index revealed that on average L. indica is more tolerant of Cd than L. fauriei. Moreover, plants in the experiment accumulated Cd differentially. In comparisons between L. indica and L. fauriei, the leaves of the former had higher concentrations of Cd, while the roots of latter had higher concentrations of Cd. Furthermore, the roots, shoots, and leaves had very high bioaccumulation factors that markedly exceeded 1.0 (exceptional only in shoots of 80 mg/kg for L. fauriei), indicating that the seedlings extracted Cd from the soil. The leaves' translocation factor of L. indica was greater than 1.0, being significantly higher than that of L. fauriei. Chlorophyll a, Chlorophyll b and total declined in both species significantly as Cd concentrations exceeded 40 mg/kg in the soil. In contrast, lipid peroxidation and proline content was found to increase with increasing Cd concentration. From the assessments of biomass production, Cd tolerance and uptake L. indica and L. fauriei could stand as excellent species for remediating Cd-contaminated soils.

Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, polyß-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ∼1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure.

Isolation and characterization of a proteinaceous α-amylase inhibitor AAI-CC5 from Streptomyces sp. CC5, and its gene cloning and expression.

An α-amylase inhibitor producing Streptomyces sp. strain CC5 was isolated from soil. A proteinaceous α-amylase inhibitor AAI-CC5 was purified from strain CC5. AAI-CC5 specifically inhibited mammalian α-amylases. The molecular weight of the inhibitor was determined to be 8,212 Da by MALDI-TOF Mass Spectrum. The N-terminal 15 amino acid residues of the purified AAI-CC5 were DTGSPAPECVEYFQS, which is dissimilar to other reported proteinaceous α-amylase inhibitors. AAI-CC5 is a pH insensitive and heat-stable protein, and cannot be hydrolysed by trypsin. AAI-CC5 was cloned and expressed in Escherichia coli BL21 (DE3) with a hexa-histidine tag on the C terminal. AAI-CC5 shared 82 % identity with Parvulustat. The recombinant α-amylase inhibitor was purified to homogeneity by one-step affinity chromatography using Ni(2+)-NTA resin with molecular mass of 9,404 Da. Steady state kinetics studies of α-amylase and the inhibitor revealed an irreversible, non-competitive inhibition mechanism with IC50 and Ki value of 6.43 ×1 10(-11) and 4.45 × 10(-11) M respectively. These results suggest this novel α-amylase inhibitor possessed powerful inhibitory activity for α-amylase, and it may be a candidate in research of diabetes therapy and obesity treatment.