DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

ERK is involved in the regulation of CpG ODN 2395 on the expression levels of anti-lipopolysaccharide factors in Chinese mitten crab, Eriocheir sinensis.

CpG oligodeoxynucleotides (ODN), as an effective adjuvant or immunopotentiator, activate the immune system and induce various immune responses. Recently, it has also been reported that high dose of CpG ODN can lead to immunosuppression. However, the underlying mechanism of CpG ODN-mediated immune response remains largely unknown in invertebrates. In the present study, the role of ERK in regulating expression levels of anti-lipopolysaccharide factors (ALFs) induced by different doses of CpG ODN 2395 was analyzed in Chinese mitten crab, Eriocheir sinensis. The mRNA expression levels of EsALFs (EsALF1, EsALF2 and EsALF3) and EsERK in haemocytes were observed to increase from 6 h to 48 h post low doses of CpG ODN 2395 (0.5 µg and 2.5 µg) stimulation, while they were suppressed after high dose of CpG ODN 2395 (12.5 µg) injection. Meanwhile, the phosphorylation levels of ERK in haemocytes were significantly promoted after low doses of CpG ODN 2395 injection, and a reduce level of ERK phosphorylation was observed after high dose of CpG ODN 2395 injection. Further investigation showed that the expression levels of EsALFs induced by CpG ODN 2395 were markedly down-regulated after knocking down the expression of EsERK. Similarly, the EsALFs mRNA expression were also inhibited post different doses of CpG ODN 2395 stimulation in PD98059 (ERK inhibitor) injection crabs. These results collectively suggest that ERK is involved in regulating the expression level of EsALFs induced by different dose of CpG ODN 2395 in Chinese mitten crab, which contribute to the understanding of the regulation of CpG ODN involving in immune response in crustaceans.

Effect of waste tire rubber crumb on impermeability of cement-stabilized soil.

To alleviate the application limitations of cement-stabilized soil in impermeable engineering and promote the recycling of waste rubber tires, crumb rubber produced from waste rubber tires was used to improve the engineering properties of the soil. The effect of crumb rubber on the permeability of the soil under different conditions was investigated using compression, compaction, and permeability tests. Crumb rubber can effectively improve the impermeability of cement-stabilized soil. The impermeability efficiency of crumb rubber is between 11% and 45%. Cleaning crumb rubber with water and Na2CO3 solution can reduce the hydraulic conductivity of rubberized cement-stabilized soil (RCSS) by 7.9%-63.6%. This, in turn, increases the unconfined compressive strength by 4.1%-25.5%. The hydraulic conductivity of the RCSS decreases with an increase in the cement content, curing duration, and void ratio. A crumb rubber concentration of 10%-20% is suitable for enhancing the impermeability of the RCSS and satisfying its strength requirements. The NOF of the equation used to predict the hydraulic conductivity of the RCSS by the rubber content, cement content, and curing duration is less than 0.35. The linear correlation between the predicted and measured values of hydraulic conductivity was determined to be 0.995 from the k-quc correlation model. The results show that the hydraulic conductivity of RCSS can be estimated reliably based on the mix ratio and compressive strength.

Seismic performance of the intermediate unit of a scrap tire-granular material composite column.

This study focused on the seismic performance of an intermediate unit of scrap tire-granular material composite columns under cyclic loading. A Laboratory test study regarding the dynamic deformation characteristics of the intermediate unit of a scrap tire-granular material composite column was conducted. The hysteresis loops, skeleton curves, stiffness degradation curves, and energy-dissipation capacities of the three specimens with different initial vertical loads were experimentally evaluated. Based on the test results, a restoring force model of the tire-granular material unit is established. The results demonstrated that the external force had a significant influence on the strength of the intermediate unit of the scrap tire-granular material composite column. The area of the hysteresis loop of the tire-granular material unit was large, indicating a good seismic performance. As the initial vertical load increased, the horizontal bearing capacity and lateral stiffness of the intermediate unit of the scrap tire-granular material composite column increased. The calculation results of the skeleton curve and hysteresis curve were in good agreement with the test results. As a base damping material, the scrap tire-granular material composite column is feasible, and provides a new concept for the resource utilization of the original scrap tire.

The involvement of hypoxia inducible factor-1α on the proportion of three types of haemocytes in Chinese mitten crab under hypoxia stress.

Hypoxia triggers diverse cell physiological processes, and the hypoxia inducible factors (HIFs) are a family of heterodimeric transcription factors that function as master regulators to respond to hypoxia in different cells. However, the knowledge about the hypoxic responses especially cell alteration mediated by HIFs under hypoxia stress is still limited in crustaceans. In the present study, a hypoxia-inducible factor-1α (HIF-1α) gene was identified (designed as EsHIF-1α). The relative mRNA expression level of EsHIF-1α was highest in hyalinocytes and lowest in granulocytes among three types of haemocytes in crabs. Hypoxia could significantly increase the EsHIF-1α protein expression level in haemocytes. Meanwhile, the proportion of hyalinocytes began to increase from 3 h post hypoxia treatment, and reached the highest level at 24 h. However, the opposite variation in proportion of granulocytes was observed under hypoxia stress. Further investigation showed that the inhibition of EsHIF-1α induced by KC7F2 (HIF-1α inhibitor) could lead to the significant decrease in the proportion of hyalinocytes under hypoxia stress, and also resulted in an increase of granulocytes proportion. While, after EsHIF-1α was activated by IOX4 (HIF-1α activator), the proportion of hyalinocytes was significantly up-regulated and the proportion of granulocytes was significantly down-regulated under post hypoxia treatment. These results collectively suggested that EsHIF-1α was involved in the regulation of proportion of three types of haemocytes induced by hypoxia stress, which provided vital insight into the understanding of the crosstalk between hypoxia and cell development in invertebrates.

PCDHB15 as a potential tumor suppressor and epigenetic biomarker for breast cancer.

Breast cancer is among the most frequently diagnosed cancer types and the leading cause of cancer-related death in women. The mortality rate of patients with breast cancer is currently increasing, perhaps due to a lack of early screening tools. In the present study, using The Cancer Genome Atlas (TCGA) breast cancer dataset (n=883), it was determined that methylation of the protocadherin ß15 (PCDHB15) promoter was higher in breast cancer samples than that in normal tissues. A negative association between promoter methylation and expression of PCDHB15 was observed in the TCGA dataset and breast cancer cell lines. In TCGA cohort, lower PCDHB15 expression was associated with shorter relapse-free survival times. Treatment with the DNA methyltransferase inhibitor restored PCDHB15 expression in a breast cancer cell line; however, overexpression of PCDHB15 was shown to suppress colony formation. PCDHB15 methylation detected in circulating cell-free DNA (cfDNA) isolated from serum samples was higher in patients with breast cancer (40.8%) compared with that in patients with benign tumors (22.4%). PCDHB15 methylation was not correlated with any clinical parameters. Taken together, PCDHB15 is a potential tumor suppressor in cases of breast cancer, which can be epigenetically silenced via promoter methylation. PCDHB15 methylation using cfDNA is a novel minimally invasive epigenetic biomarker for the diagnosis and prognosis of breast cancer.

Validated method for determination of eight banned nitroimidazole residues in natural casings by LC/MS/MS with solid-phase extraction.

A sensitive method based on solid-phase extraction-liquid chromatography-tandem mass spectrometry interfaced with electrospray ionization (SPE-LC-MS/MS-ESI) was developed for the simultaneous determination of 8 banned nitroimidazole (NOZ) drugs including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole, ornidazole, secnidazole, metronidazole-OH (MNZOH, the metabolite of MNZ), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the metabolite of RNZ and DMZ) in natural casings. After extraction with ethyl acetate and evaporation, the NOZs were reconstituted in ethyl acetate and purified on a strong cation-exchange SPE column, and then LC/MS/MS analysis was performed by positive ESI applying multiple reaction monitoring of 2 transition reactions for each compound. The method was validated according to the European Union requirements (Commission Decision 2002/657/EC). Specificity, linearity, decision limit (CCalpha), detection capability (CCbeta), accuracy, and precision were determined. Average recoveries of the 8 NOZs from natural animal casing fortified at 3 levels (0.1, 0.5, and 1 microg/kg) ranged from 87.3 to 116.5%. The calculated CCalpha for NOZs ranged from 0.029 to 0.049 microg/kg, and CCbeta ranged from 0.049 to 0.083 microg/kg. Repeatability was in the range of 3.35-10.1%, and within-laboratory reproducibility was <10.3%.

Fractal Analysis of the Fracture Evolution of Freeze-Thaw Damage to Asphalt Concrete.

AC (asphalt concrete)-13, as the main material used in pavement construction, has been applied widely in seasonal frozen areas. In order to understand the fracture mechanism in the freeze-thaw (F-T) damage process, the mesoscale structure of AC-13 is obtained by computed tomography (CT). The fractal dimension of cracks is used as a damage evaluation index. Most previous studies have only focused on the fractal dimensions of whole cracks, while ignoring the fractal tectonic process and the self-similarity degree of a single fracture. Therefore, in this study, the intrinsic mechanism of fractures and damage were investigated. In addition, the critical crack stress and fracture toughness models of a single fracture in a freeze-thaw damage process are established for AC-13. The results indicate that in terms of the critical crack stress and fracture toughness, with the increase of F-T times, there is an obvious decreasing trend. The fracture model can effectively describe the fracture toughness calculated by ABAQUS in the process of freeze-thaw cycles.

Experimental Study on the Vegetation Growing Recycled Concrete and Synergistic Effect with Plant Roots.

Vegetation growing recycled concrete (VGRC) is a relatively new building material that has both biocompatibility and engineering function. The basic performance of VGRC was investigated by experimental analysis, and the hydration products and pore structure of different VGRC mix proportions were studied by X-ray diffraction (XRD), scanning electron microscope (SEM), and industrial computed tomography (CT). The results show that ultrafine slag can reduce Ca(OH)2 content in cementing material and has a filling effect on micropores. VGRC has the best performance; the internal pore distribution is uniform when porosity is 20-25%, and the ultrafine slag content is 40%. The compressive strength of VGRC is greatly damaged by the quick-freezing method, while the degree of damage from natural freeze-thaw cycles is relatively small. Soaking in acid solution can effectively reduce the internal pore alkalinity of VGRC. Most plants can grow normally in vegetation concrete, and plant roots can penetrate 6-cm thick concrete blocks after being planted for 60 days. The compressive strength of VGRC decreased after turf planting of 30 days and then increased slowly. The permeability coefficient of VGRC increases with the increase in porosity and aggregate size and decreases after planting and covering. The frost resistance of VGRC is enhanced, and the influence of aggregate size and porosity is small after turf planting.

Effect of Freeze-Thaw Cycles on Triaxial Strength Property Damage to Cement Improved Aeolian Sand (CIAS).

Natural aeolian sand has the characteristics of low cohesion and poor water stability. In order to improve its crack resistance properties in the process of freeze-thaw cycles, P.O 42.5 ordinary Portland cement was added to form a mixture called cement improved aeolian sand (CIAS). SEM was used to analyze the microscopic micro-structure of CIAS at different times (7 days and 28 days). The mechanical properties of CIAS samples affected by freeze-thaw cycles were tested in a triaxial instrument, and gray-scale images of the three-phase distribution in the CIAS after freeze-thaw cycling were obtained by computed tomography (CT) scanning technology. The pore characteristic parameters (pore area, fractal dimension, and crack length) were studied by digital image process technique. Based on classical Griffith fracture theory, the development of the crack length and crack width with increasing freeze-thaw cycles is determined. Assuming that the pore area subordinates to the Weibull distribution, the parameters of the Weibull distribution, the damage evolution defined by the elastic modulus attenuation, and the pore area development of CIAS were determined. Research shows the cohesion decreases and internal friction angle increases with increasing cycle numbers. Three development patterns are observed: crack growth, crack closure, and crack merging, and the three patterns interact during freeze-thaw cycling. Furthermore, the fractal dimension of the pore edge fluctuates with the increasing number of freeze-thaw cycles. This work provides a theoretical basis for the application of aeolian sand and develops a method for disaster prevention in applications of freeze-thaw cycling.

Laboratory Investigation on the Shrinkage Cracking of Waste Fiber-Reinforced Recycled Aggregate Concrete.

This paper aims to study the effectiveness of adding waste polypropylene fibers into recycled aggregate concrete (RAC) on shrinkage cracking. The influences of fiber properties (length and content) on the shrinkage performance of RAC are investigated. Firstly, through the plat-ring-type shrinkage test and free shrinkage test, both of the early age and long-term shrinkage performance of waste fiber recycled concrete (WFRC) were measured. Then, X-ray industrial computed tomography (ICT) was carried out to reflect the internal porosity changes of RAC with different lengths and contents of fibers. Furthermore, the compressive strength and flexural strength tests are conducted to evaluate the mechanical performance. The test results indicated that the addition of waste fibers played an important role in improving the crack resistance performance of the investigated RAC specimens as well as controlling their shrinkage behaviour. The initial cracking time, amount and width of cracks and shrinkage rate of fiber-reinforced specimens were better than those of the non-fiber-reinforced specimen. The addition of waste fibers at a small volume fraction in recycled concrete had not obviously changed the porosity, but it changed the law of pore size distribution. Meanwhile, the addition of waste fibers had no significant effect on the compressive strength of RAC, but it enhanced the flexural strength by 43%. The specimens reinforced by 19-mm and 0.12% (volume fraction) waste fibers had the optimal performance of cracking resistance.

Determination of closantel residues in milk and animal tissues by HPLC with fluorescence detection and SPE with oasis MAX cartridges.

A liquid chromatographic method for the determination of closantel residues in milk and tissues is developed and validated. An acetonitrile-acetone solution (80:20, v/v) is used for the extraction of closantel residues from milk and animal tissues, and the extract is purified by solid-phase extraction with Oasis MAX cartridges and a mixture of formic acid-acetonitrile (5:95, v/v) as the elution solution. A C(18) bonded silica column is used for chromatographic separation. The mobile phase consists of acetonitrile-water (85:15, v/v) containing 0.05% triethylamine at pH 2.5, adjusted with phosphoric acid with the flow-rate set at 1.0 mL/min. Using the fluorescence emission of closantel at lambda(ex) = 335 nm and lambda(ex) = 510 nm, the calibration curve is linear, with a correlation coefficient of 0.9999 over the concentration range of 10-5000 microg/kg for the tissue sample and 10-5000 microg/L for the milk sample. The detection limit (s/n = 3) is 3 microg/kg for tissue sample and 3 microg/L for milk sample. The intra- and inter-day repeatabilities are between 3.35-7.66% and 4.04-8.67%, respectively. The proposed method enables the quantitative determination of closantel residues at levels as low as 10 microg/kg in animal tissue samples and 10 microg/L in milk samples.

Validated method for determination of ultra-trace closantel residues in bovine tissues and milk by solid-phase extraction and liquid chromatography-electrospray ionization-tandem mass spectrometry.

A liquid chromatographic-electrospray ionisation-tandem mass spectrometry method (LC-ESI-MS/MS) with solid extraction was developed and validated for the detection and determination of closantel residues in bovine tissues and milk. An acetonitrile-acetone mixture (80:20, v/v) was used for one-stage extraction of closantel residues in bovine tissues and milk samples, and the extract was cleaned by solid phase extraction with Oasis MAX cartridges. The mass spectrometer was operated in multiple reactions monitoring mode with negative electrospray interface. The limits of detection in different matrices were in the range of 0.008-0.009 microg/kg. The overall recoveries for bovine muscle, liver, kidney and milk samples spiked at four levels including MRL were in the range of 76.0-94.3%. The overall relative standard deviations were in the range of 3.57-8.61%. The linearity is satisfactory with a correlation coefficient (r(2)) of 0.9913-0.9987 at both concentration ranges of 0.02-100 microg/kg and 200-5000 microg/kg. The method is capable of identifying closantel residues at > or =0.02 microg/kg levels and was applied in the determination of closantel residues in animal origin foods.

Determination of banned 10 azo-dyes in hot chili products by gel permeation chromatography-liquid chromatography-electrospray ionization-tandem mass spectrometry.

An accurate method based on the use of gel permeation chromatography (GPC)-liquid chromatography-tandem mass spectrometry interfaced with electrospray ionization (GPC-LC-ESI-MS/MS) was devised for the simultaneous determination of Sudan (I-IV), Sudan Orange G, Sudan Red B, Sudan Red G, Sudan Red 7B, Butter Yellow and Para Red in hot chili products. A GPC clean-up procedure was developed for simultaneous quantification of 10 dyes in hot chili and hot chili products avoiding some interference and permitting multiple injections without damaging the column. A HPLC was performed on an Inertsil C18 column using a multistep gradient elution with 0.1% formic acid and methanol as the mobile phase. Mass spectral acquisition was done in positive ion mode. Linearity of around three orders in the magnitude of concentration was generally obtained with the correlation coefficients (r2) of 0.9984-0.9997. Limit of detection (LOD) and limit of quantification (LOQ) for the investigated dyes were in the ranges of 0.1-1.8 and 0.4-5.0 microg/kg depending on matrices, respectively. The recoveries of the 10 synthetic dyes in five matrices ranged from 81.7 to 92.9%. The intra- and inter-day precision (RSDs) was between 2.9-7.8 and 3.9-8.1%, respectively. This method has been applied successfully for the determination of the studied 10 banned dyes in hot chili products.

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction.

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.

Determination of diclazuril, toltrazuril and its two metabolites in poultry tissues and eggs by gel permeation chromatography-liquid chromatography-tandem mass spectrometry.

A new procedure has been described for the extraction of diclazuril (DIZ), toltrazuril (TOZ) and its two main metabolites toltrazuril sulphoxide (TZSO) and toltrazuril sulphone (TZS) from poultry tissues and eggs, using gel permeation chromatography (GPC). The analytes and the deuterated internal standard were extracted from the samples with ethyl acetate. The analytes were measured by LC coupled to an electrospray ionization tandem mass spectrometer operating in the negative ion mode. Excellent linear dynamic range was observed from 1 to 500 µg/L with the correlation coefficients (R(2)) better than 0.99 for all analytes. The method LOQ of the four analytes in real samples was 1.2 µg/kg for DIZ and TOZ, and 1.8 µg/kg for TZSO and TZS. These values are far lower than the maximum residue limits (MRLs) established by several control authorities. The developed method was accurate with overall recoveries in four matrices.

[Simultaneous determination of residues of six imidazolinone herbicides in adzuki beans by high performance liquid chromatography-electrospray ion trap tandem mass spectrometry].

A method for the simultaneous determination of six pesticides in adzuki beans by high performance liquid chromatography-electrospray ion trap tandem mass spectrometry was developed and evaluated. The sample was extracted with 0.1 mol/L NH4HCO3 (pH 5)-methanol (70 : 30, v/v). The extract was partitioned with dichloromethane, and cleaned up with gel permeation chromatography. The separation was performed on an Inertsil ODS-3 column (150 mm x 2.1 mm, 5 microm) with the gradient elution of methanol and 1% acetic acid as mobile phase. The identification and quantification were performed by electrospray ion trap mass spectrometry with selected ion monitoring mode. The calibration curves of imidazolinone herbicides showed good linearities in the range of 10 - 200 microg/L (5 -100 microg/L for imazaquin) with the correlation coefficients between 0.998 7 and 0.999 7. The limits of detection ranged between 0.2 microg/kg and 0.5 microg/kg (S/N = 3). The average recoveries of six imidazolinone herbicides spiked in adzuki beans ranged between 81.6% and 99.4% (n = 6) with the relative standard deviations of 3.1% -7.8%. The method has been used for the determination of 6 imidazolinone herbicides simultaneously in adzuki beans with easy operation, high accuracy and good precision.