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Secondary organic aerosol (SOA) has been identified as a major contributor to fine particulate matter (PM2.5) in the North China Plain (NCP). However, the chemical mechanisms involved are still unclear due to incomplete understanding of its multiple formation processes. Here we report field observations in summer in Handan of the NCP, based on high-resolution online measurements. Our results reveal the formation of SOA via photochemistry and two types of aqueous-phase chemistry, the latter of which include nocturnal and daytime processing. The photochemical pathway is the most important under high-Ox (i.e., O3 + NO2) conditions (65.1 ± 20.4 ppb). The efficient SOA formation from photochemistry (Ox-initiated SOA) dominated the daytime (65 % to OA), with an average growth rate of 0.8 µgm-3h-1. During the high-relative-humidity (RH; 83.7 ± 12.5 %) period, strong nocturnal aqueous-phase SOA formation (aqSOA) played a significant role in SOA production (45 % to OA), with a nighttime growth rate of 0.6 µgm-3h-1. Meanwhile, an equally fast growth rate of 0.6 µgm-3h-1 of Ox-initiated SOA from daytime aqueous-phase photochemistry was also observed, which contributed 39 % to OA, showing that photochemistry in the aqueous phase is also a non-negligible pathway in summer. The primary-related SOA (SOA attributed to primary particulate organics) and aqSOA are related to residential coal combustion activities, supported by distinct fragments from polycyclic aromatic hydrocarbons (PAHs). Moreover, the conversion and rapid oxidation of primary-related SOA to aqSOA were possible in the aqueous phase under high-RH conditions. This work sheds light on the multiple formation pathways of SOA in ambient air of complex pollution and improves our understanding of ambient SOA formation and aging in summer with high oxidation capacity.

Secondary organic aerosol (SOA) has been identified as a major contributor to fine particulate matter (PM2.5) in North China Plain (NCP). However, the chemical mechanisms involved are still unclear due to incomplete understanding of its multiple formation processes. Here we report field observations in summer in Handan of NCP, based on high-resolution online measurements. Our results reveal the formation of SOA via photochemistry and two types of aqueous-phase chemistry, the latter of which include nocturnal and daytime processing. The photochemical pathway is the most important under high Ox (=O3 + NO2) conditions (65.1 ± 20.4 ppb). The efficient SOA formation from photochemistry (phochem-SOA) dominated the daytime (65 % to OA) with an average growth rate of 0.8 μg m−3 h−1. During the high relative humidity (RH: 83.7 ± 12.5 %) period, strong nocturnal aqueous-phase SOA formation (aq-SOA) played a significant role in SOA production (45 % to OA) with a nighttime growth rate of 0.6 μg m-3 h-1. Meanwhile, an equally fast growth rate of 0.6 μg m-3 h-1 of phochem-SOA from daytime aqueous-phase photochemistry was also observed, which contributed 39 % to OA, showing that photochemistry in the aqueous phase is also a non-negligible pathway in summer. The primary-related-SOA (SOA attributed to primary particulate organics) and aq-SOA are related to residential coal combustion activities, supported by distinct fragments from polycyclic aromatic hydrocarbons (PAHs). Moreover, the conversion and rapidly oxidation of primary-related-SOA to aq-SOA could be possible in the aqueous phase under high-RH conditions. This work sheds light on the multiple formation pathways of SOA in ambient air of complex pollution, and improves our understanding of ambient SOA formation and aging in summer with high oxidation capacity.

Current research is focused on the development of drug candidates from natural products. Rhein a Traditional Chinese Medicine (TCM) from Polygonaceae (rhubarb) has exhibited antioxidant, anti-inflammatory and anticancer activities, however no work has reported its antiviral potential, thus this study was performed to investigate the antiviral activities of rhein on new castle disease virus (NDV) in vitro.NDV infection of chicken embryo fibroblasts (CEFs) was prepared using 10-day-old specific pathogen free chicken embryos. Cytotoxicity and anti-viral activities of rhein were assessed using the MTT method. The interaction between NDV and cell membrane proteins were also detected using virus overlay protein binding assay (VOPBA). In addition NDV genes expressions in CEFs were measured using real-time fluorescent quantitative (RTFQ) PCR.The results showed that rhein effectively inhibit NDV activities maximal safe concentration of 0.125 mg/ml. This finding indicated that, rhein could be used as future antiviral drug against NDV.[Formula: see text].

Non-methane hydrocarbons (NMHCs) plays an important role in haze events. This study focused on NMHCs characteristics, effects on secondary organic aerosol (SOA) and source apportionment in Handan in heavily polluted seasons, i.e., autumn (October 2017) and winter (January 2018) and their differences in non-haze and haze days. An online instrument (AMA GC5000) was used to monitor NMHCs in Handan firstly at the campus of Hebei University of Engineering. It was found that the average NMHCs concentrations were 41.45 and 57.56 ppb in autumn and winter, respectively, which were higher than most investigated domestic and foreign megacities to the best of our knowledge. Alkanes was the largest contributor to NMHCs (45.4%), and followed by alkenes, aromatics, and acetylene. In haze days, the concentrations of NMHCs and four categories increased far more in winter than those in autumn compared to non-haze days. The total SOA potential (SOAP) in winter (3364 μg m-3) was higher than that in autumn (2804 μg m-3). The total SOAP was close in haze and non-haze days in autumn, while it was much higher in haze days in winter. Regardless of the variations of different periods, aromatics contributed most to the total SOAP, with contributions of 87 - 95%, where toluene was the most contributed species. Combined the results of diagnostic ratios with positive matrix factorization (PMF) model, NMHCs of Handan was mainly contributed by traffic emissions and combustion events. Vehicle related (from 23.2 to 30.1%) and industrial related source (from 13.7 to 26.7%) may be major contributors to the haze pollution in autumn and winter, respectively.

Volatile organic compounds (VOCs) are important precursors in the formation of ozone (O3) and secondary organic aerosol (SOA). Hence, using an online monitor, we measured the concentrations of 56 VOC species in Handan, one of the most polluted cities in China, from Sep. 2017 till Aug. 2018. We also characterized the VOC pollution by measuring the O3 and NOx and determining the chemical reactivity of the VOCs at different levels of O3 pollution, analyzing the regional O3 formation mechanisms, and apportioning the VOC sources. The daily VOC concentrations displayed a wide range, from 37.7 to 288.9 µg m–3, with an average of 112.0 ± 45.5 µg m–3, during the observation period, and the contribution of alkenes and other aromatics to the O3 formation potential (OFP) of the VOCs was higher on medium pollution days than clean days. Furthermore, the majority of the O3 during spring and winter was formed in VOC-sensitive regimes, whereas the high diurnal concentrations during summer and autumn were produced in NOx-sensitive regimes. Based on the i-butane/n-butane and i-propane/n-propane ratios, the main sources of butane and propane were vehicular exhaust and liquid gasoline, respectively. Source apportionment by principal component analysis (PCA) revealed that the VOCs in Handan originated from vehicular emission, industrial production, solvent use, gasoline evaporation, and combustion.

China implemented the National Air Pollution Prevention and Control Action Plan since 2013, aiming at significantly improving air quality in major areas and cities in 2017. To investigate the changes of the characteristics of PM2.5 and its major chemical components, formation mechanisms, and sources apportionment during 2013–2017 in urban Handan, which was one of the top polluted cities in China, we analyzed the chemical compositions of PM2.5 samples for the entire year of 2013 and 2017, as well as a comprehensive dataset including continuous online hourly observations of the meteorological parameters and major air pollutants, i.e., SO2, NO2, and O3 in this study. Potential Source Contribution function (PSCF) analysis was applied to investigate the potential source regions contributing to high PM2.5 pollution episodes. The results showed that the concentration of major air pollutants except ozone decreased, and the proportion of PM2.5 decline in Handan (37.0%) had reached the requirements (25%) of the National Air Pollution Prevention and Control Action Plan. The major chemical compositions of PM2.5 decreased significantly, but the percentage of secondary organic aerosols increased from 37.0% in 2013 to 40.0% in 2017. The higher proportion of secondary components implied more severe secondary pollution. The PSCF analysis indicated that the significant local contribution and short-range transport in the surrounding areas were the major sources of heavy pollution episodes in Handan. This study highlights the importance of emission reduction of atmospheric organic compounds and joint control actions on a regional basis during heavy pollution episodes.

Handan has been one of the most polluted cities in China since 2013 and became the top city for PM2.5 in 2017. In this research, we observed coarse particulate matter (PM10), fine particulate matter (PM2.5), submicron particulate matter (PM1), and the chemical composition of PM2.5 from November 16, 2015, till March 14, 2016, in Handan. During the observation period, hourly concentrations of PM10, PM2.5, and PM1 peaked at 1070.1, 864.4, and 519.5 µg m–3, respectively. Severe pollution occurred on a large fraction of days in the heating season, which was characterized by frequent and long-lasting pollution episodes. A large fraction of the transport trajectories during nine typical episodes during that period in Handan were from the northwest. Water-soluble ions (sulfate, nitrate, and ammonium) in PM2.5 accounted for the largest proportion at all pollution levels. The highest proportion of SIA occurred in a heavily polluted episode, during which it was as high as 50.0% (sulfate: 18.8%, nitrate: 18.7%, and ammonium: 12.5%). The sulfate and ammonium in PM2.5 increased gradually while the nitrate decreased as the level of pollution rose from clean to heavily polluted. The fraction of SOA and OM to PM2.5 decreased as the pollution level increased, indicating a weakening of photochemical reactions. The POA in PM2.5 increased with the aggravation of haze, and the heterogeneous chemistry was enhanced by the aggravation of pollution. Liquid reactions were important in the formation of sulfate during pollution and non-pollution stages. Liquid reactions of NO2 are enhanced in the pollution stage during the heating season in Handan.

This study aimed to investigate the thermal-oxidative aging and virgin-old recycling behavior of SBS polymer modified asphalt. For the same, the four-component method, the Fourier transform infrared spectroscopy (FTIR), and the gel permeation chromatography (GPC) test were adopted for characterizing five types of asphalt binders. Besides, Brookfield rotary viscosity and time scan tests were conducted for measuring the rheological parameters of the asphalt samples. The results indicated the following: (i) Thermal-oxidative aging deteriorated the fluidity and performance stability of the asphalt. The aromatic ring index, IC=C, was able to reflect the change in the temperature sensitivity of the asphalt during the process of aging. (ii) After thermal oxidation, the increase in viscosity was mainly attributed to the increase of asphalt macromolecules weight. A reduction in the proportion of the dispersion medium in the asphalt colloid structure was observed to be the main reason for its decreased fatigue resistance potential. (iii) By adding fresh asphalt, the virgin-old recycling approach slowed the thermal-oxidative aging, primarily due to physical integration. (iv) In this study, the asphalt viscosity-temperature properties were more sensitive to molecular changes than fatigue properties.

In this paper, the method of vibration immersion erosion was designed to simulate the sodium salt erosion of bitumen, and the modified dynamic shear rheology (DSR) tests were applied to explore the self-healing ability and fatigue life of bitumen in different sodium salt erosion environments. Moreover, the main reasons for the bitumen performance change were explained from the microscopic level by the four-component test, Fourier-transform infra-red (FT-IR) spectroscopy, and scanning electron microscopy (SEM). The significance ranking of different influencing factors (salt solution concentration, erosion time, and temperature) was determined by the F-test, and the correlation between the self-healing ability and fatigue life of the bitumen in salt erosion environments was analyzed. It was found that the self-healing ability and fatigue life of the bitumen were severely weakened in sodium salt solutions. Na2SO4 had the most prominent weakening effect, followed by CH3COONa and NaCl. The microscopic analysis revealed that the colloidal balance of the bitumen was destroyed under sodium salt erosion and the interaction between sodium salts and some radical groups of the bitumen resulted in the deterioration of bitumen fluidity, stability, and mechanical properties, resulting in the decline of the self-healing ability and fatigue life of the bitumen. The rankings of different influencing factors on the self-healing ability and fatigue life of the bitumen were temperature > concentration > time and temperature > time > concentration, respectively. The correlation analysis expressed that the negative impacts of sodium salts on the self-healing ability and fatigue life of the bitumen were highly synchronized.

To comprehensively evaluate the transport pathways and regional sources of PM2.5 in the “2 + 26” cities, air quality simulations using WRF-Chem were pursued over January, April, July, October 2015. The seasonal characteristics, vertical distributions of PM2.5 fluxes, and transport pathways were investigated. The model reasonably predicted the meteorological parameters (e.g., T2, Q2, WS10, and WD10) and air pollutants in both evaluated domains. The horizontal PM2.5 fluxes were strong in October (1221 kt, −1222 kt) and April (1119 kt, −1142 kt) and weak in January (933 kt, −997 kt) and July (589 kt, −809 kt). The directions of horizontal flux contributors relative to their target cities were entirely different in January and July. The same pattern can also be found at vertical layers. However, there was no uniform direction seen in April and October. A total of 14 pathways at the boundaries of the “2 + 26” cities were identified. The SW-NE pathways, which were dominant in Beijing and Tianjin with flux contributions of 65% and 75%, were found in all four months. The NW-SE pathways, which were the major pathways of Taiyuan (87%), and the W-E pathways, which were the major pathways of Zhengzhou (44%) and Jinan (43%), always showed different directions in January and July, following the directions of prevailing winter and summer monsoon winds. Moreover, Shijiazhuang (42%) and Handan (48%) were dominated by S–N pathways, which showed different patterns throughout the four months. The results could be helpful for implementing policies aimed at ecological compensation and joint air pollution control.

Beijing, the capital of China, is a mega city with a population of >20 million. In recent years, the city has experienced heavy air pollution, with particulate matter (PM) being one of its top pollutants. In the last decade, extensive efforts have been made to characterize the sources, properties, and processes of PM in Beijing. Despite progress made by previous studies, there are still some important questions to be answered and addressed. The focus of this research is to study the impact of the heterogeneous hydrolysis of N2O5 on the formation of nitrate (NO3-) and ammonium (NH4+) in Beijing. The results show that during heavy pollution days (e.g., during 14-17 September 2015, with PM2.5 concentration over 100μg/m3), the concentrations of NO2 and O3 were high, with maxima of 90 and 240μg/m3, respectively, providing high precursors for the formation of N2O5. In addition, the aerosol and sulfate concentrations were also high, with maxima of 201μg/m3 and 23μg/m3 respectively, providing reacting surface for the heterogeneous reaction. As a result, the hydrolysis of N2O5 led to 21.0% enhancement of nitrate (NO3-) and 7.5% enhancement of ammonium (NH4+). It is worth to note that this important effect only occurred in high pollution days (PM2.5 concentration over 100μg/m3). During low-pollution periods (PM2.5 concentration

The focus of this study is to evaluate the impact of biomass burning (BB) from South Asia and Southeast Asia on the glaciers over the Tibetan Plateau. The seasonality and long-term trend of biomass fires measured by Terra and Aqua satellite data from 2010 to 2016 are used in this study. The analysis shows that the biomass burnings were widely dispersed in the continental of Indian and Southeast Asia and existed a strong seasonal variation. The biomass burnings in winter (January) were relatively weak and scattered and were significantly enhanced in spring (April). The highest biomass burnings located in two regions. One was along the foothill of Himalayas, where is a dense population area, and the second located in Southeast Asia. Because these two high biomass burning regions are close to the Tibetan Plateau, they could have important effects on the BC deposition over the glaciers of the Tibetan Plateau. In order to study the effect of BB emissions on the deposition over the glaciers in the Tibetan Plateau, a regional chemical model (WRF-Chem; Weather Research and Forecasting Chemical model) was applied to simulate the BC distributions and the transport from BB emission regions to the glaciers in Tibetan Plateau. The result shows that in winter (January), due to the relatively weak BB emissions, the effect of BB emissions on BC concentrations was not significant. The BC concentrations resulted from BB emissions ranged from 0.1 to 2.0 μg/m3, with high concentrations distributed along the foothill of Himalayas and the southeastern Asia region. Due to the relative low BC concentrations, there was insignificant effect of BB emissions on the deposition over the glaciers in the Tibetan Plateau in winter. However, the BB emissions were highest in spring (April), producing high BC concentrations. For example, along the Himalayas Mountain and in the southeastern Asia region, The BC concentrations ranged from 2.0 to 6.0 μg/m3. In addition to the high BC concentrations, there were also west and south prevailing winds in these regions. As a result, the BC particles were transported to the glaciers in the Tibetan Plateau, causing significant deposition of BC particles on the snow surface of the glaciers. This study suggests that the biomass burning emissions have important effects on the BC deposition over the glaciers in the Tibetan Plateau, and the contaminations of glaciers could have significant impact on the melting of snow in the Tibetan Plateau, causing some severe environmental problems, such as the water resources.

The purpose of this study was to investigate the anti-Newcastle disease virus (NDV) activities of baicalin from Scutellaria baicalensis, a Traditional Chinese Medicine in vitro. Chicken embryo fibroblasts (CEFs) were infected with NDV, and quantitative analysis of apoptotic cells was performed using flow cytometry. Cytotoxicity and anti-viral activities of baicalin were studied using the MTT method. The results showed that the maximal safe concentrations of baicalin to CEFs was 1 × 2(-2) mg/ml. Baicalin could directly kill NDV, inhibit the infectivity of NDV to CEF and block intracellular NDV. It inhibited the apoptosis of NDV-infected CEFs and suppressed the spread of NDV. These results indicate that baicalin has strong anti-NDV activity and has the potential for use as components of an antiviral drug.

The purpose of this study was to investigate the anti-Newcastle disease virus (NDV) activities of 9-oxo-10,11-dehydroageraphorone (euptox A) from Eupatorium adenophorum Spreng (E. adenophorum) in vitro. NDV infection of chicken embryo fibroblasts (CEFs) was performed. Cytotoxicities and antiviral activities of euptox A was assessed by the MTT method. The interaction of NDV with cell membrane protein was detected by virus overlay protein binding assay (VOPBA). The expression levels of NDV genes in CEFs was tested by RTFQ PCR. The results showed that the maximal safe concentrations of euptox A to CEFs was 10 μg/mL. Euptox A could directly neutralise NDV, inhibit the infectivity of NDV to CEFs and block intracellular NDV treat NDV infection. And euptox A brings competitiveness inhibition for NDV binding to its receptors and then prevent NDV infection. These results indicated that euptox A possessed anti-NDV activity has potential use as components of a natural antiviral drug.

Euptox A (9-oxo-10, 11-dehydroageraphorone), the main toxin isolated from Eupatorium adenophorum, is known to induce immunotoxicity in animals. However, the precise mechanism underlying the effects of Euptox A on splenocytes is unclear. Here, we aimed to investigate the molecular mechanisms underlying the effect of Euptox A in mouse spleens after its intragastric administration and found that Euptox A exhibits proautophagic effects in splenocytes. Euptox A markedly arrested the splenocytes in the G0/G1 phase, which was accompanied by inhibition of the expression of the positive regulators CDK4, CDK2, cyclin D1, PCNA, and E2F1, and promotion of the expression of the negative regulators p53, p21 Waf1/Cip1, p27 Kip1, and Chk1. We also found that Euptox A did not markedly induce splenocyte apoptosis, but induced autophagy while increasing the subcellular localization of punctate LC3, ratio of LC3-II/LC3-I, and Beclin 1 levels, and decreasing p62 levels. Euptox A also significantly inhibited p-PI3K, p-p38 MAPK, p-Akt, and p-mTOR expression, but increased PTEN and p-AMPK expression. These results indicated that Euptox A induced splenocyte autophagy by inhibiting the PI3K/Akt/mTOR pathway, suppressing p38 MAPK expression, and activating AMPK. These findings provide new insights into the mechanisms involved in spleen toxicity caused by Euptox A in mice.

Beijing, the capital of China, is a mega city with a population of20 million. In recent years, the city has experienced heavy air pollution, with particulate matter (PM) being one of its top pollutants. In the last decade, extensive efforts have been made to characterize the sources, properties, and processes of PM in Beijing. Despite progress made by previous studies, there are still some important questions to be answered and addressed. The focus of this research is to study the impact of the heterogeneous hydrolysis of N

To reduce the immunogenicity of recombinant staphylokinase, structure-based mutagenesis of Glu80 residue in wild-type staphylokinase (wt-Sak) was rationally designed and carried out by a modified QuikChange site-directed mutagenesis. Sak mutants, including Sak(E80A) and Sak(E80S), were successfully expressed in E. coli DH5a as a soluble cytoplasmic proteins and accounted for more than 40% of the total cellular proteins. The expressed proteins were purified by a three-step chromatographic purification process. SDS-PAGE and HPLC analyses results indicated that the purified proteins were almost completely homogeneous and the purities of Sak mutants exceeded 97%. Analysis of fibrinolytic activity revealed that substitution of E80 residue with serine and alanine resulted in slightly increased specific activities of Sak mutants. Investigation of the immunogenicity of Sak mutants showed that the amount of specific anti-Sak IgG antibodies elicited by Sak(E80A) and Sak(E80S) in BALB/c mice decreased approximately 35% and 27%, respectively compared with wt-Sak. The abilities of Sak mutants to stimulate proliferation of T cells from BALB/c mice and to bind mouse anti-Sak polyclonal serum were significantly lower than those of wt-Sak. These results suggested that substitution of Glu80 residue by alanine and serine successfully eliminated part of T- and B-cell epitope of Sak molecule. Our findings suggested that simultaneous elimination of T- and B-cell epitopes was a useful method to reduce the immunogenicity of wt-Sak molecule and provided a strategy for engineering safe Sak-based fibrinolytics for the clinical treatment of acute myocardial infarction.

Five years measurements were used to evaluate the effect of emission controls on the changes of air pollutants in Beijing and its surroundings in the NCP during 2008 Olympic Games (2008OG). The major challenge of this study was to filter out the effect of variability of meteorological conditions, when compared the air pollutants during the game to non-game period. We used four-year (2007, 2009–2011) average as the Non-2008OG to smooth the temporal variability caused by meteorological parameters. To study the spatial variability and regional transport, 6 sites (urban, rural, a mega city, a heavy industrial city, and a remote site) were selected. The result showed that the annually meteorological variability was significantly reduced. Such as, in BJ the differences between 2008OG and 5-years averaged values were 2.7% for relative humidity and 0.6% for wind speed. As a result, the anomaly of air pollutants between 2008OG and Non-2008OG can largely attribute to the emission control. The comparison showed that the major pollutants (PM10, PM2.5, NO, NOx) at the 6 sites in 2008OG were consistently lowered. For example, PM2.5 in BJ decreased from 75 to 45 μg/m3 (40% reduction). However, the emission controls had minor effect on O3 concentrations (1% reduction). In contrast, the O3 precursor (NOx) reduced from 19.7 to 13.2 ppb (33% reduction). The in-sensitivity between NOx and O3 suggested that the O3 formation was under VOCs control condition in NCP, showing that strong VOC emission control is needed in order to significantly reduce O3 concentration in the region.

The purpose of this study was to investigate the anti-Newcastle disease virus (NDV) activities of baicalin from Scutellaria baicalensis, a Traditional Chinese Medicine in vitro. Chicken embryo fibroblasts (CEFs) were infected with NDV, and quantitative analysis of apoptotic cells was performed using flow cytometry. Cytotoxicity and anti-viral activities of baicalin were studied using the MTT method. The results showed that the maximal safe concentrations of baicalin to CEFs was 1 × 2(-2) mg/ml. Baicalin could directly kill NDV, inhibit the infectivity of NDV to CEF and block intracellular NDV. It inhibited the apoptosis of NDV-infected CEFs and suppressed the spread of NDV. These results indicate that baicalin has strong anti-NDV activity and has the potential for use as components of an antiviral drug.

E. adenophorum has reported to cause hepatotoxicity. But, the precise effects of E. adenophorum on hepatocytes is unclear. Saanen goats were fed on E. adenophorum to detect the cytotoxicity effects of E. adenophorum on hepatocytes. Our study has shown that the typical apoptotic features, the increasing apoptotic hepatocytes and activated caspase-9, -3 and the subsequent cleavage of PARP indicated the potent pro-apoptotic effects of E. adenophorum. Moreover, the translocation of Bax and Cyt c between mitochondria and cytosol triggering the forming of apoptosome proved that the mitochondria-mediated apoptosis was triggered by E. adenophorum. Furthermore, E. adenophorum increased the MDC-positive autophagic vacuoles and the subcellular localization of punctate LC3, the ratio of LC3-II/LC3-I and the protein levels of Beclin 1, but decreased that of P62, indicating the potent pro-autophagic effects of E. adenophorum. In addition, E. adenophorum significantly inhibited the protein leves of p-PI3K, p-Akt and p-mTORC1, but increased PTEN and p-AMPK. Also, the p-mTORC2 and p-Akt Ser473 were inhibited, indicating that the supression of mTORC2/Akt pathway could induce the autophagy of hepatocytes. The autophagy-realted results indicated that the inhibition of PI3K/Akt/mTORC1- and mTORC2/Akt-mediated pathways contributed to the pro-autophagic activity of E. adenophorum. These findings provide new insights to understand the mechanisms involved in E. adenophorum-caused hepatotoxicity of Saanen goat.

The precise cytotoxicity of E. Adenophorum in relation to the cell cycle and apoptosis of splenocytes in Saanen goats remains unclear. In the present study, 16 Saanen goats were randomly divided into four groups, which were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The results of TUNEL, DAPI and AO/EB staining, flow cytometry analysis and DNA fragmentation assays showed that E. adenophorum induced typical apoptotic features in splenocytes, suppressed splenocyte viability and caused cell cycle arrest in a dose-dependent manner. However, westernblot, ELISA, qRT-PCR and caspase activity analyses showed that E. adenophoruminhibited Bcl-2 expression, promoted Bax translocation to the mitochondria, triggered the release of Cytc from the mitochondria into the cytosol and activated caspase-9 and -3 and the subsequent cleavage of PARP. Moreover, in E. adenophorum-induced apoptosis, the protein levels of Fas, Bid, FasL and caspase-8 showed no significant changes. E. adenophorum treatment induced the collapse of ΔΨm. Moreover, these data suggested that E. adenophorum induces splenocyte apoptosis via the activation of the mitochondrial apoptosis pathway in splenocytes. These findings provide new insights into the mechanisms underlying the effects of E. adenophorum cytotoxicity on splenocytes.

To reduce immunogenicity of recombined staphylokinase (r-Sak) , site-directed mutagenesis of Arg77 and Glu80 residue was performed to simultaneously remove T and B cell epitope in r-Sak molecule.The solvent accessible surface areas of residues 77 and 80 in r-Sak were used to analyze rational design of Sak mutation. The Sak mutants were expressed in E coli DH5alpha. After purified by a 3-step chromatography, their fibrinolytic activities and immunological properties were analyzed.Immunogenicity tests suggested that Sak induced a Th2-type immune response. Substitution of Glu80 with alanine or serine successfully reduced its solvent accessible surface area while simultaneously removing part of the T and B cell epitope. Changing Arg77 to glutamine, asparagine, or lysine removed only part of the T cell epitope. Of six dually substituted variants, Sak (R77Q/E80A) and Sak(R77Q/E80S) variants effectively eliminated part of the B and T cell epitopes, which markedly reduced their immunogenicity.

To develop a more potent thrombolytic agent, four Sak (staphylokinase) variants were constructed, in which RGD (Arg-Gly-Asp) sequences are introduced into diferent sites of the N-terminus of Sak. These variants were successfully expressed in Escherichia coli DH5alpha as soluble cytoplasmic proteins in a 5-litre fermentor and accounted for more than 40% of the total cellular protein. The expressed proteins were subsequently purified, employing a similar three-step chromatographic purification process. SDS/PAGE and HPLC-MS analyses indicated that the purified proteins were almost completely homogeneous, the purity of the variants exceeding 95%. Further investigations into the properties of the Sak variants showed that mutations at the N-terminus significantly affected N-terminal methionine excision, and serine residues at the N-terminus of Sak appeared to play an important role in the process. Kinetic analysis of r-Sak (recombinant Sak) and its variants using plasminogen as substrate indicated that the mutations affected the proteolysis. In addition, a significant inhibitory effect of the Sak variants at 2.0 muM was observed on the ADP-induced aggregation of platelets compared with that of r-Sak, whether N-terminally cleaved or not (P

To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive L: -arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of L: -arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.