Due to the different research areas and research periods, the existing analyses of the correlation between PM2.5 and O3 have reached different conclusions. In order to clarify the relationship between PM2.5 and O3, this study selected mainland China as the research area, based on the PM2.5 and O3 concentration data of 1458 air quality monitoring stations, and analyzed the correlation between PM2.5 and O3 for different time scales and geographic divisions. Moreover, by combining the characteristics of the pollutants, topography, and climatic features of the study area, we attempted to discuss the causes of the spatial and temporal differences of R-PO (the correlation between PM2.5 and O3). The study found that: (1) R-PO tends to show a positive correlation in summer and a negative correlation in winter, (2) the correlation coefficient of PM2.5 and O3 is lower in the morning and higher in the afternoon, and (3) R-PO also shows significant spatial differences, including north–south differences and coastland–inland differences.In recent years, particulate matter of 2.5 µm or less (PM The Ministry of Ecology and Environment of the People’s Republic of China concluded in the “2016 China Environmental Status Bulletin” that among the 74 major cities in China, the number of days with particulate matter of 2.5 µm or less  [3 is a strong oxidizing substance formed by photochemical reaction [3 is a kind of photochemical pollutant and can cause potential harm to human health [3 are nitrogen oxides (NOx) and VOCs [3.Both PMn health . In addis (VOCs) ,7,8. O3 reaction ,10. Whatn health ,12,13,14and VOCs ,16,17. U2.5 concentration has been controlled, to a certain extent. However, the concentration of O3 has not decreased, but has in fact increased [2.5 decreased by 12.3% between 2013 and 2015. In addition, Li et al. [2.5, PM10, sulfur dioxide (SO2), carbon monoxide (CO), and nitrogen dioxide (NO2) all decreased from 2014 to 2016 but, at the same time, the annual mean concentration of O3 increased by more than 10 µg/m3. The above research results show that although the non-attainment rate of PM2.5 is still high in most parts of the country [3, whose concentration has been increasing year on year, has become the major pollutant in most parts of China [2.5 and O3 have already caused a huge threat to national health [In order to cope with the serious air pollution problems, the Chinese government has implemented a series of atmospheric emission control policies in recent years, so that PMncreased ,19. Guo ncreased  noted thi et al.  reported country ,23, its of China . As a nel health ,25.2.5 and O3 that they both have common precursors, i.e., VOCs. It is therefore possible that there is a certain relationship between their pollution conditions. Harden [2.5 and O3 concentrations and their relationship when analyzing the air conditions in the Great Smoky Mountains National Park in the United States and found that these two pollutants showed obvious positive correlations in every season from January 2008 to December 2011. Most scholars that have addressed this issue have taken individual regions of China as the research areas. In the process of analyzing the pollution mechanism of the research areas, the correlation between PM2.5 and O3 (which we term “R-PO” in this paper) has been determined. The research results showed that, in Beijing , the R-PO is positive in summer. In the Changsha-Zhuzhou-Xiangtan district , the R-PO is positive in summer, negative in winter, and is mainly negatively correlated during other periods [3 and five other types of conventional pollutants (including PM2.5) in Jiangsu Province, then found that the correlation differed between the warm and cold seasons. The results showed that the annual R-PO coefficient was less than 0.2, in the hot season it was less than 0.4, and in the cold season it was weak or showed a negative correlation. Wang et al. [2.5 and O3 had a strong positive correlation in summer, a negative correlation in winter, and an obvious spatial difference between three regions under the same time. Although the research area was nationwide, the authors did not pay close attention to the correlation between PM2.5 and O3. In conclusion, PM2.5 and O3 have different relationships in different regions and during different time periods.Although the process of air pollutant formation and conversion is very complicated, and the direct relationship between photochemical smog and particulate pollutants is difficult to quantify, it can be seen from the chemical reaction mechanism of PM. Harden  analyzed periods ,28. Chen periods  analyzedg et al.  analyzed2.5 and O3, it can be seen that the existing air pollution studies have not regarded R-PO as the research focus. The research related to the relationship between the two pollutants has shown great differences in research areas, time scales, and research methods, so that different conclusions have been drawn under the influence of complex topography, meteorological conditions, and pollutant emissions. In addition, due to the limited space-time characterization of R-PO, there is not enough analytical basis to carry out in-depth analysis and systematic summary of the relationship between the two pollutants. Li et al. [3 because of stronger solar radiation. The simulation results with the Goddard Earth Observing System Chemical Transport Model (GEOS-Chem) by Li et al. [2.5 over the 2013–2017 period, which slow down the aerosol sink of hydroperoxy (HO2) radicals and thus stimulate ozone production. Therefore, to obtain a systematical and explicit relationship between PM2.5 and O3 which is not limited to a certain area, it is necessary to describe the spatial-temporal characteristics of R-PO in detail.From the above studies investigating the relationship between PMi et al.  speculati et al.  indicate2.5 and O3 monitoring data of all the ground monitoring stations in mainland China, with the research period from March 2013 to February 2017. The temporal characteristics and spatial distributions of each stations’ R-PO are described in the daily mean scale and hourly scale respectively, according to the seasonal divisions. Considering the spatial-temporal characteristics of R-PO, the causes of the R-PO patterns are further discussed with regard to the physical properties and chemical mechanisms of the pollutants and the climatic and meteorological factors. Through the analysis process of this paper, we can comprehensively and systematically gain an understanding of the spatial and temporal differences of R-PO and the possible influencing factors from a macroscopic perspective, to provide statistical evidence for the fine-scale study of the chemical mechanisms and physical properties, and the influence of meteorological factors on pollutants. We hope that the results of this study will help people to better understand the relationship between PM2.5 and O3 and provide a reference for the possible inclusion of this variable in the retrieval models of other parties.This study is based on PMThe research area of this study—mainland China—is located in the southeast of Eurasia, on the west coast of the Pacific, with a total area of 9.6 million square kilometers. This vast area has complex topography. In addition, the unique geographical location and complex terrain create complex climatic features in mainland China, i.e., the summer is hot and rainy, the winter is cold and less rainy, and the temperature and rainfall vary greatly between the north and the south. This geographical and climatic complexity adds more variability and heterogeneity to China’s air pollution situation.2.5 and O3 hourly monitoring data with a high temporal resolution for the mainland of China , which were obtained from the National Air Quality Monitoring Network, from 1 March 2013, to 28 February 2017. The spatial distribution of the latest air quality monitoring stations is shown in http://106.37.208.233:20035/).In this study, we used PM2.5 and O3 monitoring data were preprocessed. The observation values which were abnormally high or congregated unreasonably were removed . The preprocessed results showed that the number of effective sites nationwide on 1 March 2013, was 494, distributed in 74 cities, which gradually increased to 1544 by 28 February 2017, distributed in 367 cities across the country, covering all prefecture-level administrative regions in mainland China. It is worth mentioning that, to ensure the reliability of the daily average concentration, the daily average data were used only if the effective hours of the original observation data were greater than 18 h.Since there are a few abnormalities in the raw observation data, the hourly PMChina has a vast territory spanning five time zones, but the air monitoring data released by the National Air Quality Monitoring Network uses unified Beijing time . In order to get a more accurate diurnal variation, we made local time corrections for each site. The optimization rules are shown in 2.5 and O3, this study was based not only on the PM2.5 and O3 daily mean concentration at each site, but also the hourly data. Taking the seasonal characteristics of the pollutants into account, the data for each site were divided into season-based units . In this study, we choose the “Pearson” correlation coefficients to quantify the relevance of PM2.5 and O3, which was widely used in the research about the relations between different pollutants. The correlation coefficient between PM2.5 and O3 calculated in Pearson’s way is abbreviated as R-PO. To be specific, R-PO is obtained for each station, one by one, for the PM2.5 and O3 data pairs in each season over the four years, about 360 days, and the significance level (p) values were also calculated at the same time . Accompanied by a p-value test, we could get a reliable correlation result of more than 1500 stations. The detailed data organization process is shown in In order to make full use of the existing data, and to understand the relevance of PMThere are a few points to note: (1) R-PO was first calculated one by one within each site, and (2) when it is necessary to summarize multiple stations, the average processing in the whole country or a certain region was adopted according to the geographical location.According to the spatial-temporal features of R-PO, the causes of this characterization were then further discussed from two aspects: (1) the influence of the physical properties and the chemical mechanisms of pollutants, and (2) the effect of climatic and meteorological factors on R-PO. For facilitating a clearer and more systematic conclusion, the national situation was also summarized in the research process. Considering the different climatic characteristics and geographical locations of each region, the country was divided into seven regions: Northwest China, Southwest China, Northeast China, North China, East China, Central China, and South China  and the relationship between O3 and these factors, are the opposite. Then, in addition to the negative correlation between PM2.5 concentration and O3 concentration in long time series due to meteorological factors, is there still a deeper connection within each season? In the vast area of China, what kind of spatial heterogeneity will each site show? The analytical studies described in the following sections were conducted so as to further understand the correlation of them.Based on the research of relevant scholars ,32,33,34g et al.  pointed 2.5 and O3 is biased towards positive and in winter, it tends to be negative.The spatial distribution of R-PO in the 1458 stations nationwide, in each season, is shown in 2.5 and O3 concentrations across the country and the corresponding R-PO are plotted in Based on the hourly data, the diurnal change of the PM2.5, O3 concentration, and R-PO in the four seasons are similar, only differing at the numerical level, i.e., there are higher concentrations of O3, lower concentrations of PM2.5, and higher R-PO values in summer, and there are lower O3 concentrations, higher PM2.5 concentrations, and lower R-PO values in winter.It can be seen from 2.5 peaks at around 10:00, and then gradually decreases, reaching the lowest value of the day around 16:00, and then slowly rises, reaching another peak value at around 22:00, and then maintaining a high concentration at night. The diurnal change of O3 concentration is relatively simple compared to PM2.5, and is single-peak/single-valley, i.e., the lowest value is around 8:00, gradually rising to around 15:00 when the peak value is reached. It is worth noting that the time of the minimum R-PO (Rmin) value coincides with the peak time of PM2.5, i.e., around 10:00, while the time of the maximum R-PO (Rmax) value is consistent with the peak time of O3, i.e., around 16:00.Based on the diurnal variation curve, it can be observed that PMIn addition, it can be found from the Rmax and Rmin values in 2.5 and O3 concentrations show opposite trends, not only in the diurnal variation curve, but also in the daily mean concentration curve. By correlating R-PO with the concentration of both pollutants, it can be found that R-PO tends to be positive when O3 is higher and PM2.5 is lower, and negative when PM2.5 is higher and O3 is lower.Comparing 2.5 and O3. The physical properties of PM2.5 and O3 and the chemical mechanisms associated with them may also have an impact on their correlation. In addition, according to the spatial heterogeneity of R-PO in the different seasons and at different times, it can be considered that the climatic differences in the different regions and the meteorological changes over the course of a day may also be related to R-PO. In the following, we provide a cause discussion of the spatio-temporal representation of R-PO from these two aspects.Therefore, based on the above R-PO patterns in the different seasons and times of the day, it is speculated that the value of R-PO may have a certain relationship with the concentration of PM2.5 and O3 respectively, of the same position, in the corresponding seasons. The experimental results were used to analyze whether the value of R-PO has a certain correlation with the concentration of PM2.5 and O3, so as to better link the experimental results with the pollution mechanisms. In addition, to clarify the difference in the R-PO values of different regions, a distinction strategy was made between the regions to which the sites belong. It can be observed from 2.5 concentration and R-PO show different distribution characteristics in the different seasons. In summer, the concentration of PM2.5 is lower, and R-PO values are densely distributed in the positive correlation area. The PM2.5 concentration in spring and autumn is slightly higher than that in summer, and the distribution of R-PO is more dispersed than in summer. The concentration of PM2.5 is higher in most areas in winter, and the R-PO values are more concentrated in the negative correlation area. Observing the distribution of the scatter points in each season, it can be seen that the scatter plots of the four seasons are all distributed in a triangular shape. This means that as the PM2.5 concentration becomes higher, the value of R-PO shows a slight decrease. In other words, the higher the PM2.5 concentration is, the more the R-PO tends to be negative.In order to further analyze the influencing factors of R-PO, we start with the relationship between R-PO at all the stations and the average concentrations of PM2.5, an aerosol with an aerodynamic diameter of less than 2.5 µm, it can reflect the concentration level of total aerosol in the atmosphere, to a certain extent. Studies have also shown that the PM2.5 concentration is strongly positively correlated with the extinction coefficient [2.5, meaning a higher aerosol content in the atmosphere, the extinction effect of the particulate matter and the light absorption effect of the carbon-containing aerosol mixing process both have a great influence on the illumination. In addition, light intensity is an indispensable condition for O3 generation [2.5 concentrations in winter and around 10:00 of each day.Based on the research findings on the mechanisms of air pollution, it is known that the light extinction coefficient of particulate matter which can absorb light is very high in some areas, at up to 91.9% . As one fficient . Therefoneration ,39. This2.5 concentration, the extinction effect caused by the absorption and reflection of light by the particles and the light absorption effect during the internal mixing of the particulate components may together cause the negative R-PO.In summary, it can be generalized that with high PM2.5, the seasonal variation of R-PO with O3 concentration is not obvious. Combined with the seasonal characteristics and diurnal variations of R-PO, R-PO tends to be positive when the O3 concentration is higher in spring and summer and at 16:00 and is inclined to be negative when the concentration is lower in autumn and winter and around 10:00. Since the change of R-PO with O3 concentration is not obvious, it can be speculated that the positive tendency of R-PO may be related to the formation process of O3. The regional differences depicted in Compared with PM2.5 is very high, and the main components of secondary organic aerosols (SOAs) are sulfate, nitrate, and ammonium salt, formed by condensation and chemical reaction of SO2, NOx, NH3, and VOCs [3, but also generate the main components of SOAs at the same time, thus increasing the concentration of PM2.5, to a certain extent. Griffin et al. [3 are generated by VOCs through photo oxidation and the gaseous/particle equalization process. Shao et al. [3 and secondary particulate matter, resulting in a composite superposition of high concentrations of O3 and PM2.5 in the atmosphere. In addition, Guo et al. [3 resulting in R-PO positive bias, many studies have shown that the oxidation of O3 can promote the conversion of primary gaseous pollutants into particulate pollutants. The research of Wonaschütz et al. [3 concentration. This is because the high concentration of O3 makes the atmospheric environment highly oxidizing, so that gaseous pollutants such as sulfur dioxide and nitrogen oxide in the air are more easily oxidized and converted into fine particles such as sulfates and nitrates, thereby causing PM2.5 concentration to increase.It is known that the proportion of secondary components in PMand VOCs . The conand VOCs ,42. Zhanand VOCs  noted thn et al.  reportedo et al.  observedo et al.  stated tz et al.  indicate3 simultaneously, and the oxidation of O3 can promote gas/particle conversion, under the combined effect of these two factors, PM2.5 and O3 possibly tend to show a positive correlation in summer and around 16:00, with the strong light and high temperature. Due to the hysteresis of the secondary reaction, O3 usually peaks around 16:00, and the peak of R-PO also appears at 16:00, instead of 14:00, when there is the most intense light. Therefore, it is possible that the promotion effect of O3 oxidation influencing PM2.5 generation on R-PO may be greater than the effect of the photochemical reaction causing simultaneous generation of PM2.5 and O3 on R-PO.Based on the above related explanation, it is reasonable to summarize the mechanism as follows. Because the photochemical reaction can generate PM and O2.5 and O3. Therefore, meteorological factors are bound to affect R-PO by influencing the concentrations of PM2.5 and O3 under the effect of the physical properties and chemical mechanisms of the pollutants described in As we all know, there is a high correlation between the climatic characteristics of different regions and the meteorological changes over a short period of time and the concentration of PM2.5 pollution. Besides, the low temperature of winter causes the mixed gases from car engines to burn at a slower pace, which leads to incomplete combustion and an increase of motor vehicle exhaust emissions, thus causing an increase of PM2.5. Tai et al. [2.5 is likely to remain high. For O3, the high temperature and strong radiation in summer help to increase O3 concentration. The effect of PM2.5 and O3 concentration levels on R-PO is detailed in Due to the low temperature in the north during winter, the heating of coal leads to an increase of sulfur dioxide and dust particles in the air, which greatly increases the PMi et al.  and Ji ei et al.  both not2.5 and O3 concentration, while PM2.5 and O3 concentration would make an influence on R-PO (see In a word, the climatic characteristics of the different regions and the meteorological changes in a short period of time could affect the PMBecause of the latitude difference caused by the north–south span, the different distances from the sea caused by the east–west span, and the differences in topographical terrain, the different regions of China have various climatic characteristics. Northeast China, North China, East China, Central China, and South China are vulnerable to the monsoons caused by the difference in sea and land temperature, due to their close proximity to the ocean. This is the reason why, for weather characterized by monsoon climate, rainfall and heat usually occur at the same time. The temperature and rainfall gradually increase from the north to the south of China. The northwestern region far from the ocean does not receive many humid air masses, so it is characterized by a dry, less rainy temperate continental climate. In the southwestern region, the high-altitude Qinghai–Tibet Plateau features a plateau/mountain climate with low temperature throughout the year, which is cold and dry with long sunshine duration and strong solar radiation.3 also promotes PM production, which was described in Combined with the seasonal variation and the spatial distribution map of R-PO in 2.5 and O3, resulting in the R-PO being biased to a negative value. This is also the reason why 2.5 levels.What is more, the strongly positive R-PO of coastal areas and the southeast monsoons from the ocean in summer are consistent. As we all know, monsoons from the ocean can bring clean air and abundant precipitation. The cleansing effect of the monsoons may promote the positive correlation of R-PO. Some scholars believed that air masses from the ocean, relatively stable wind speed and wind direction, and strong solar radiation, all facilitate the conversion of gaseous pollutants into new particles ,50. Acco2.5 and O3 always show a positive correlation in the afternoon.Based on the above conclusions, it can be speculated that the diurnal variation of R-PO is also related to the changes of meteorological conditions, such as light intensity, wind speed, relative humidity, and the atmospheric boundary layer. Driven by solar radiation, meteorological conditions show significant changes throughout the day. With the gradual strengthening of solar radiation after sunrise, the wind speed increases with the increase of temperature, and the boundary layer rises. The wind speed always reaches a maximum at around 14:00 and, at the same time, the boundary layer is the highest and the light intensity is the strongest ,52,53,543 concentration, the above analysis results indicate that the main reasons for the high value of R-PO in summer and every afternoon and in coastal and low-latitude areas are strong solar radiation, the cleansing effect of monsoons, and rainfall from the ocean. To find out to what extent the monsoons and rain affect the intensity of light, thereby promoting the positive bias of R-PO, further studies on the interaction between these meteorological factors are needed.Although the positive value of R-PO is not directly related to the O2.5 and O3 concentration data for all the monitoring stations of the China Air Quality Monitoring Network, this paper has presented the spatial and temporal characteristics of the correlation between PM2.5 and O3. The results show that PM2.5 and O3 tend to be positively correlated in summer and negatively correlated in winter. In addition, the correlation coefficient between PM2.5 and O3 is lower at around 10:00, while the value is higher at around 16:00. The correlation between PM2.5 and O3 also shows obvious spatial differences, including a north–south difference and a coastal–inland difference. Specifically, there is a positive correlation in the south and a negative correlation in the north. Coastal areas tend to be positively correlated, and inland areas tend to be negatively correlated.By using PM2.5 and O3 are usually produced with the discharge of the primary pollutant, because of their secondary pollutant characteristics. Under this circumstance, the two pollutants usually show a positive correlation. This is why the correlation coefficient of individual regions in the north during spring, autumn, and winter can rise in the morning between 6:00 and 8:00. When the meteorological conditions are more favorable for the formation of PM2.5, the two pollutants show a negative correlation, and the higher the PM2.5 concentration is, the more strongly it inhibits the formation of O3, because of PM2.5′s extinction characteristic. When meteorological conditions are more favorable for the generation of O3, the two pollutants are positively correlated under the influence of photochemical reaction and atmospheric oxidation. Therefore, it can be postulated that the decrease of PM2.5 concentration in recent years has reduced its influence on atmospheric light intensity, thus indirectly promoting the production of O3, which demonstrates the simulation result that the decrease of PM2.5 concentration has made a certain impact on the O3 trend proposed by Li et al. [According to the spatial and temporal characterizations of R-PO, we can speculate that the level of R-PO is affected by the physical characteristics of the pollutants, the chemical mechanism of air pollution, the climatic differences between regions and seasons, and the meteorological factors in a short period of time. Summarizing all the experimental and analytical conclusions obtained in this paper, we try to speculate the mechanism of R-PO as follows. When humans undertake activities such as industry and travel and emit primary air pollutants, PMi et al. . The mec3 replacing PM2.5 as the primary pollutant in summer is mainly due to the emission conditions of its precursors and the meteorological conditions. Controlling the emission of primary pollutants should be the top priority in the process of environmental air quality control.However, the phenomenon of O


After two stages of immunizations, the average potency in plasma of all groups reached more than 1,000 IU / mL except that in group 4. In group 5, the first two basic immunizations with TT and the subsequent immunizations with TeNT-Hc, it showed slightly higher antibody titers and potency. This study demonstrated that TeNT-Hc is a safe, effective, and yet easy-to-produce low-cost immunogen and suitable for TT replacement in tetanus antitoxin production.The demand of tetanus antitoxin (TAT) as tetanus treatment in developing and underdeveloped countries is still great since it is relatively easy to achieve and affordable. However, there are still issues in the preparation of highly effective TAT with tetanus toxoid (TT) as the immunogen. The tetanus toxin native C-fragment (TeNT-Hc) retains many properties and it is a very promising candidate for the development of tetanus human vaccine. In this study, we tested the immunogenicity of TeNT-Hc in the preparation of tetanus antibodies, by TeNT-Hc alone or in different combinations with TT. The antibody titers and components in horse serum or plasma in different groups were analyzed and compared with those immunized by the conventional TT and it showed comparability with the results of traditional methods. The plasma efficacy and Clostridium tetani (C. tetani) infection that causes tetanus toxin production ) can be determined.According to the method in Chinese Pharmacopoeia , different volumes (100 Lf/mL) of tetanus toxin standard solution were precisely-measured and added to the corresponding reaction tubes. Then diluted horse plasma (1 mL) was added quickly into those tubes and mixed thoroughly. The tubes were immersed in a water bath (45-50°C) and observed closely, and the volume of standard solution for first occurrence of flocculation was recorded. After repeating three times, the horse plasma efficacy (in flocculation unit (Lf/mL) =ca 0.5 IU/mL) and different concentrations of horse plasma (diluted by borate buffer) were mixed with 0.2mL of tetanus toxin standard, respectively. The mixtures were incubated at 37°C for 1 h and were then injected into mice intraperitoneally. There were three mice in each group and each group was observed at least twice a day for the first five days. The control group should all die within 72 to 120 hours. The efficacy of the horse serum was evaluated as the highest dilution which is most likely to die of the same symptoms as the control mice.According to the method in Chinese Pharmacopoeia , 0.2mL of the tetanus antitoxin standard  values <0.05 were considered to be significant and marked as ∗. P<0.01 was considered to be very significant and marked as ∗∗.Unpaired two-tailed Student'sAs shown in p≥0.05) of anti-TT IgG antibody titers in serum among the groups except group 4 one week after the first, second, and third immunization. The lowest anti-TT IgG antibody titer in group 4 has significant difference with the highest antibody titer in group 5 (p <0.05). Likewise, there were no significant difference in anti-TeNT-Hc IgG antibody titer, anti-TT, and TeNT-Hc IgM antibody titers between groups observed in immunization progress (p> 0.05). All immune methods can induce humoral immunity based on IgG antibody.Serum titers of anti-TT, anti-TeNT-Hc IgG or IgM induced by TeNT-Hc alone or TeNT-Hc and TT co-immunization were tested. As shown in p <0.05). However, there was no significant difference of anti-TT IgG titers among the groups after the second and the third stage immunization (p≥0.05). The titer of anti-TeNT-Hc IgG antibody in group 4 was slightly lower and significantly lower than those in group 5 after the first and third stage immunization (p<0.05). No significant difference of anti-TeNT-Hc IgG titers was found among the other groups (p≥0.05). The anti-TT and anti-TeNT-Hc IgG titers in plasma after three stages of immunization with TeNT-Hc were compared with those collected after routine immunization with TT. The levels of anti-TT IgG antibodies among different groups had no significant difference (p≥0.05). However, the titers of anti-TeNT-Hc IgG antibody in the plasma of the TT conventional immunized group were significantly lower than those of the six groups of horse plasma immunized with TeNT-Hc (p <0.01).In addition to serum antibodies, antibody titers of anti-TT and anti-TeNT-Hc IgG in horse plasma one week after each stage of immunization were also studied . As the p<0.05). There was no significant difference in antibody titers among groups after the first and the third stage of immunization (p≥0.05).The titers of antitoxins specific binding to TeNT-Hc in different groups of horse plasma were evaluated of at different immunization stages by an agarose diffusion method. According to the result shown in p <0.05). There was no significant difference among groups 1, 2, 3, 5, and 6 (p≥0.05).In our preliminary experiments, the potency of TeNT-Hc with a protein concentration of 0.17 mg / mL was determined to be equivalent to the titer of tetanus toxoid (TT) of 100 Lf / mL. The potency of each group after three immunization stages was compared by using the TeNT-Hc (0.17 mg / mL) as the antigen. As shown in  in vivo neutralization test in mice was used to test the antitoxin potency of the plasma in each group one week after each stage of immunization. As shown in p≥0.05).The E. coli, has good immunogenicity, is low-cost, is easy to scale up, and is purified, not mention it is completely safe and nontoxic to both humans and horses. TeNT-Hc as a candidate for human tetanus vaccine is currently well underway and sued as a potential replacement of TT in TAT production. In this paper, using an established immunization procedures and doses, the in vivo immunogenicity of TeNT-Hc in horses was tested alone or in combination with TT antigen. The antibody titers in serum or plasma in different groups were compared with those immunized by the conventional TT and it showed very similar results from those using TT immunization. After additional two stages of immunization, the average potency in plasma of all groups reached more than 1,000 IU / mL except that in group 4. With refining the immunization methods and adjust the dosage, we have confidence that the results could easily exceed the potency required for tetanus antitoxin production. The purpose of this study is to evaluate the possibility of TeNT-Hc as an immunogen to replace TT in the tetanus antitoxin production. The difference in immune programs will affect the titers of antibodies and antitoxins. The immunization program used in this study is a routine and optimized procedure for the preparation of tetanus antitoxin by TT immunization. For TeNT-Hc, this program may be not the optimal program and it is necessary to further explore the best immunization program in the follow-up study.As an effective immunogen, TT has played a critical role in the preparation of TAT. However, there were still inherent drawbacks of using TT: the production scale of toxin and bacterial strains were limited due to safety concern, the formaldehyde detoxification is generally incomplete, and the large-scale waste could cause environmental issues; the repeated immunization with considerable high-dose to the blood-harvesting horses will, sooner or later, induce the liver toxicity, resulting in reduced product quality and increased production costs. TeNT-Hc, the nontoxic fragment from TT and recombinant expressed inAs the experimental results show, there was no significant difference between the groups. In group 5, the first two basic immunizations with TT and the subsequent immunization with TeNT-Hc, it showed slightly higher antibody titers and potency. In group 4, which utilized the sequence of hyperimmunization with TT and the rest immunization with TeNT-Hc, it resulted in lower antibody titers and potency. The result that there was no significant difference in immunogenicity between the different dose groups and the different antigen combination groups may indicate that the lowest dose of TeNT-Hc used in the experiment was sufficient for the preparation of antitoxin. However, due to the small number of samples and large variations between the individual horses, a larger scale experiment is warranted to figure out the optimal immunization program. By optimizing the immunization schedule and the corresponding doses, TeNT-Hc may even show better immunogenicity.Without the accumulated liver toxicity from TT immunization, TeNT-Hc positions itself as a completely nontoxic recombinant replacement while retaining all the effectiveness of TT, as a long-term safety study could easily reveal. In conclusion, this study further confirmed the validity of using TeNT-Hc to be replaced or used in conjugation with TT to produce TAT.


L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces -THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.Previous studies have utilized monoamine oxidase (MAO) and  Bioproduction of tetrahydropapaveroline (THP) is limited by the specificity of monoamine oxidase (MAO). Here, the authors identify an insect 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) that can bypass MAO for direct aldehyde production and demonstrate bifunctional switching of DHPAAS for efficient THP production. Engineered metabolic systems are now being designed for production of biofuels, polymers, bulk chemicals, and pharmaceuticals2. Computational methods to select the best metabolic pathways from thousands of possibilities have been demonstrated for production of 1,4-butanediol4. The combination of metabolic engineering with enzyme engineering can even be used to construct artificial biosynthetic pathways to compounds including 2,4-dihydroxybutyric acid5.Recent progress in synthetic biology and metabolic engineering offers potential to optimize the bioproduction of virtually any compound6. Microbial production of pharmaceutical alkaloids offers advantages in terms of cost efficiency, environmental sustainability, and process control7. Recently, bioproduction of opioid alkaloids has been achieved in yeast via a norcoclaurine (higenamine) containing pathway9 and in Escherichia coli via a tetrahydropapaveroline  containing pathway11. The THP pathway requires one less enzymatic step and has afforded the highest BIA titer of 1 mM THP (287 mg/L). However, current THP bioproduction relies upon monoamine oxidase (MAO), a membrane bound flavoenzyme that is active towards many monoamines in addition to dopamine13. It is therefore desirable to engineer a soluble enzyme to improve production of THP, and downstream alkaloids including thebaine, which has been recently reported with microbial titers lower than 10 mg per litre11.With the goal of further developing enzyme engineering as a practical approach that can expand the scope of bioproduction targets, benzylisoquinoline alkaloid (BIA) biosynthesis is presented as an example for synthetic enzyme development. Opioid analgesics are derived from BIAs and are essential medicines for pain and palliative care as defined by the World Health Organization14. Specifically, a 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified from Bombyx mori and engineered to switch between two distinct activities for the direct production of THP in a symmetrical pathway. Existing reports have applied substrate specificity engineering to metabolic engineering5. In contrast, the current study applies functional enzyme engineering to the assembly of an alternative bioproduction pathway.Utilizing BIA bioproduction as a model pathway, the current system aims to search for alternative enzyme engineering targets and learn alternative biosynthetic pathways. This synthetic biology workflow includes enzyme selection and learning for synthetic pathway design via the recently developed M-path prediction software14. M-path uses an iterative random algorithm to score chemical similarities and can be operated as a web-based tool. In contrast to searching known enzyme networks, M-path is advantageous in that it can predict unknown enzymatic reactions based upon substrate and product similarities. Furthermore, M-path can find reactions and pathways from a wide range of search space and easily expand the search space14. To explore alternative BIA production pathways from aromatic amino acids, the M-path search algorithm was tested. A combined database of updated enzyme entries from BRENDA (BRaunschweig ENzyme DAtabase)15 and Kyoto Encyclopedia of Genes and Genomes (KEGG)16 was used to increase enzyme targets. Aromatic aldehyde synthase (AAS) and DHPAAS were identified as putative shortcuts for production of 4-hydroxyphenylacetaldehyde (4-HPAA or 4-HPA) from L-tyrosine (Tyr), and 3,4-dihydroxyphenylacetaldehyde  from L-3,4-dihydroxyphenylalanine (L-DOPA)  results in symmetrical BIA bioproduction pathways that are distinct from the reported MAO-mediated pathways 19, have been referred to as AAS. A more recently discovered enzyme from insects, DHPAAS (EC 4.1.1.107), catalyzes the oxidative decarboxylation of L-DOPA to produce DHPAA20, and this enzyme might therefore be considered as an AAS related protein. Phylogenetic analysis suggests that AAS and DHPAAS diverged from aromatic amino acid decarboxylase . Accordingly AAS, DHPAAS and AAAD are structurally similar and are all dependent upon pyridoxal 5′-phosphate (PLP) cofactor. Although AAS and DHPAAS were first assigned as EC 4.1.1.- by KEGG, many questions still remain about these recently characterized enzymes, which are not straightforward to classify due to bifunctional activities. Accordingly, EC 4.1.1.107–109 were only recently added to comprehensive enzymes databases like BRENDA, and rules for the selection of AAS or DHPAAS for bioproduction applications should be clearly established.AAS and DHPAAS are recently established bifunctional enzymes, which catalyze the simultaneous decarboxylation and amino group oxidation of aromatic amino acids. Two enzymes discovered in plants, phenylacetaldehyde synthase L-DOPA relative to the specificity of MAO for dopamine. Mathematical models and numerical simulations were employed to compare the production of THP via the nonsymmetrical and symmetrical pathways , which was modeled based on the structure of Sus Scrofa DDC in complex with carbidopa (PDB ID: 1JS3)24, contains a leucine residue at the position corresponding to AAAD active site His192 25 as the template. The structure of this 320–350-loop region, which participates in cross-dimer active site formation and substrate binding, improved when using a template of human histidine decarboxylase in complex with histidine methyl ester (PDB ID: 4E1O)26.Uncharacterized Lepidoptera DHPAAS comprises the central phylogenetic group Fig. , and wasB. mori and D. melanogaster DHPAAS numbering) determines catalytic activity as that of a decarboxylase or aldehyde synthase , Tyr79-Tyr80 is conserved. Lepidoptera and formicidae DHPAAS groups contain a mixture of Phe79-Tyr80, Tyr79-Tyr80 and Tyr79-Phe80.Careful comparison of DDC and DHPAAS structures suggests that DHPAAS residues Phe79 and Tyr80 play an additional role in distinguishing DHPAAS activity from DDC activity Figs . Tyr79-PB. mori sequence XM_004930959.2 was selected as a distinct DHPAAS sequence containing all three identified DHPAAS specific residues Phe79, Tyr80 and Asn192, as well as Gly353, which is reported to increase substrate specificity to L-DOPA26. In addition, Phe79Tyr, Tyr80Phe and Asn192His DHPAAS catalytic variants were designed to explore tuning the bifunctional production of dopamine and DHPAA.In this study, the B. mori XM_004930959.2 was synthesized and cloned into the E. coli expression vector pE-SUMO. DHPAAS was purified by metal affinity chromatography according to the methods section. DHPAAS activity was screened by separation of substrates and products using thin-layer chromatography (TLC). Enzymatic products were verified by LC-MS operated in multiple reaction monitoring (MRM) mode.The native sequence of wild-type B. mori XM_004930959.2 protein produced DHPAA as the major product of L-DOPA, as indicated by the identification of negative ion m/z 151.10 and lack of major dopamine ions. The identification B. mori DHPAAS indicates that the above analysis of DHPAAS phylogenetic groups is accurate. Structural analysis supports a hypothesis that the Phe79Tyr-Tyr80Phe-Asn192His triple variant would have DDC-like activity, while Asn192His and Phe79Tyr-Ty80Phe variants would result in a mixture of DHPAAS and DDC activities. To test this hypothesis, and gain insight into a more comprehensive DHPAAS mechanism, the enzymatic activities of Phe79Tyr, Tyr80Phe, and Asn192His DHPAAS variants were evaluated .After confirming that THP could be directly produced by Phe79Tyr-Tyr80Phe-Asn192His DHPAAS, in vitro production was quantified using all four designed ode Fig. . THP was2O2 mediated oxidation. After supplementation with 2.5 mM sodium ascorbate, conversion of L-DOPA to THP increased to 23% (219 µM) using Phe79Tyr-Tyr80Phe-Asn192His DHPAAS. This surpasses the highest bioconversion of dopamine to THP reported at 15.9%10. Ascorbic acid did not inhibit DHPAA production by DHPAAS indicating that DHPAA is a direct enzymatic product of L-DOPA, rather than a secondary product of dopamine oxidation by H2O2.In vitro THP production improved significantly after supplementation with ascorbate to overcome product degradation via HAs predicted, overall DHPAA production was highest with wild-type DHPAAS and the Phe79Tyr-Tyr80Phe variant, and lowest with the Asn192His and Phe79Tyr-Tyr80Phe-Asn192His variants Fig. , Table 1E. coli grown in lysogeny broth (LB) medium resulted in low THP titers, with higher amounts produced by the Phe79Tyr-Tyr80Phe variant, followed by that of wild-type DHPAAS. After changing the growth medium from LB to M9 minimal medium, THP production increased -THP was produced by DHPAAS in vivo as demonstrated by chiral LC-MS analysis -THP to reticuline under the conditions tested , accompanied by a huge increase in dopamine levels. However, reticuline titers did not simply increase dependent on THP concentration, with the bottleneck appearing strongest at the 6-OMT and 4-OMT steps. Expression of DHPAAS-Phe79Tyr-Tyr80Phe-Asn192His together with wild-type DHPAAS, as well as Coptis japonica 6-OMT, CNMT, and 4-OMT, resulted in a moderate increase in relative THP. Yet, the bottleneck to reticuline still remained, although it appeared to be relaxed at the 6-OMT and 4-OMT steps.To improve the limited reticuline titers, additional R,S)-THP to reticuline may not be optimal during simultaneous conversion of L-DOPA to THP due to disruption of pathways relating to SAM cofactor recycling -THP titer to 9.45 µM (2.71 mg/L) in the first step via wild-type DHPAAS and DHPAAS-Phe79Tyr-Tyr80Phe-Asn192His. A second addition of BL21(DE3) containing 4-OMT from C. japonica together with 6-OMT and CNMT from P. somniferum was then able to relieve the bottleneck to produce 3.7 μM 3-hydroxycoclaurine (3HC), 1.4 μM 3-hydroxy-N-methylcoclaurine (3HNMC) and 1.5 μM reticuline after an additional 18.5 h  cofactor29. To investigate the incorporation of oxygen in the DHPAAS mechanism, DHPAAS reactions were performed in D2O and H218O; however, DHPAAS activity could not be observed under these conditions. The many uncertainties about the DHPAAS and AAS mechanisms should be clarified in future studies.To scale up THP and reticuline production, improved synthetic biology design can be driven by two types of learning information: enzyme parameters and metabolomics data. Regarding learning for enzyme improvement, Phe79, Tyr80, and Asn192 were all elucidated as key residues that can tune the DHPAAS mechanism for optimized THP precursor ratios. Yet questions still remain about the exact function of these residues, including their role in OL-DOPA was high, with no observed activity for tyrosine or phenylalanine by the Phe79Tyr-Tyr80Phe-Asn192His variant. This indicates that competitive inhibition by aromatic amino acids is not a major consideration for DHPAAS-mediated biosynthesis. However, preliminary analysis indicates possible feedback inhibition of DHPAAS by dopamine and DHPAA, and possible substrate inhibition at high concentrations of L-DOPA. These factors, together with the low measured Km of B. mori DHPAAS, may help to explain why L-DOPA was not consumed during in vivo bioproduction experiments. It is also possible that DHPAAS is inhibited by unknown growth medium components. Therefore, substrate concentration, substrate specificity and inhibition should be comprehensively characterized as data for enzyme learning.DHPAAS specificity for During in vitro production of THP, high concentration dopamine was best, as more dopamine can quickly consume reactive DHPAA while it is being produced. Thus, Asn192His and Phe79Tyr-Tyr80Phe-Asn192His variants were observed to produce the highest THP levels in vitro. However, in vivo environments include many more cellular and chemical components that can compete with dopamine for reaction with DHPAA. Accordingly, the use of minimal medium together with higher levels of DHPAA produced by wild-type DHPAAS and the Phe79Tyr-Tyr80Phe variant resulted in better in vivo THP titers. These results highlight that the optimal dopamine to DHPAA ratio for THP production depends on the culture conditions.10. MAO-mediated conversion of dopamine to THP was 15.9%, without reported metrics for conversion of L-DOPA to dopamine10. In the current study, DHPAAS expressing cells produced 9.45 μM THP (2.71 mg/L) with a yield of 1.89% from 1 mM L-DOPA. The lower titer and yield may be explained by the 12.6-fold lower substrate concentration and the additional decarboxylation step from L-DOPA. Moreover, a majority of 1 mM L-DOPA remained unconsumed in the culture medium, suggesting low substrate permeability and possibly higher intracellular yields.The best reported THP titer of 1 mM (287 mg/L) was produced from 12.6 mM dopamine using MAO expressing cellsL-DOPA utilization by DHPAAS is much better in vitro, with 23% conversion of L-DOPA to 219 µM THP. In the MAO-mediated system, a THP titer of 99.2 µM resulted in the highest reticuline titers11. Therefore, in vitro results suggest that DHPAAS-mediated THP production is capable of reaching optimal levels for downstream BIA production. However, DHPAAS-mediated in vivo production needs to be improved to reach the 1 mM THP benchmark and computational yields greater than 50%.30. Indeed, the complete pathway is now assembled into a single E. coli cell to produce high titer reticuline from glucose31, 10 years after the development of the first MAO-mediated reticuline producing system32. To better match this benchmark, a complete DHPAAS-mediated pathway from glucose to (S)-reticuline is now under development. Metabolomics-directed learning data should include flux from toxic intermediates DHPAA20 and THP10 towards less-toxic reticuline. Addition of NCS can increase conversion of DHPAA to selectively produce (S)-THP, leading to better substrate conversion by SAM dependent BIA methyltranferases33. A related OMT bottleneck with impaired SAM cofactor recycling was reported for vanillin biosynthesis in E. coli grown on M9 minimal medium34. This supports balancing of SAM recycling pathways as another strategy to improve reticuline production  should also be pursued to increase BIA titers.The DHPAAS-mediated pathway also requires improvements in downstream BIA production. After the establishment of proof-of-principle bioproducers, it usually requires more than five years of optimization to reach industrial demands35, homovanillic acid, imidazole-4-acetaldehyde, and 4-amino-phenylacetaldehyde. In conclusion, this study demonstrates that functional enzyme engineering can be effectively applied to high value bioproduction pathways. Most importantly, further development of the DHPAAS-mediated pathway is required to improve downstream BIA production in future studies.Additional M-path analysis was performed to search alternative bioproduction pathways that might be improved using DHPAAS or AAS, linking the learning process of enzyme database improvement to new cycles of pathway design. For example, M-path identified EC 4.2.1.107–109 as enzymes to mediate biosynthesis of 2’-norberbamunine14. M-path scores are calculated as a Tanimoto co-efficient. The 2016 version of the M-path database was updated using the most recent substrate, product, and enzyme information from KEGG and BRENDA. Curated mode was used to search for enzymes to mediate tyrosine (PubChem CID: 6057) to THP (CID: 18519), tyrosine to 4-HPAA (CID: 440113), L-DOPA (CID: 6047) to THP, L-DOPA to DHPAA (CID: 119219), tyrosine to 2′-norberbamunine (CID: 441063), histidine (CID: 6274) to imidazole-4-acetaldehyde (CID: 150841), and 4-aminophenylalanine (CID: 151001) to 4-aminophenylacetaldehyde (CID: 20440863). For conversion of tyrosine to homovanillic acid (CID: 1738), M-path search was performed in original mode.Enzyme searches were performed using the M-path web-based version according to the methods of Araki et al.MAO13. Models were constructed with and without product feedback inhibition factored into the reaction velocities of MAO (VMAO), DDC (VDDC) and DHPAAS (VDHPAAS). Drain of DHPAA was later included in some models. Possible ranges of parameter values were surveyed and collected from literature and BRENDA15. In order to predict the performance of each pathway, parameter values were randomly generated within the set ranges, and Monte–Carlo simulation was carried out according to L-DOPA was fed as a constant term based on randomly generated parameters. When the maximum amount of 100 mM L-DOPA was reached, substrate feeding to the system was stopped. A homemade program was implemented in Python 3.0 using scipy.integrate.odeint as the solver for numerical simulations. More detailed computational methods are described in Mathematical models were constructed for the nonsymmetrical (DDC-MAO) and symmetrical (DDC-DHPAAS) pathways. For the nonsymmetrical pathway, since MAO recognizes a variety of amines, competitive inhibition was introduced into the MAO reaction velocity VB. mori DHPAAS and P. somniferum TyDC1 were generated with MODELLER22 operated within Chimera23. Crystal structures of D. melanogaster DDC (PDB ID: 3K40)25 and histidine decarboxylase (4E1O)26 were used as templates for B. mori DHPAAS modeling. The structure of Sus Scrofa DDC in complex with carbidopa (PDB ID: 1JS3)24 was used as a template for TyDC1. Binding of PLP covalently linked to aromatic amino acid substrate, and structure refinement were performed with Molecular Operating Environment (MOE). Completed structures were analyzed in PyMOL.Dimeric homology models of For phylogenetic analysis, insect AAAD and DHPAAS sequences were collected from the protein BLAST non-redundant database after searching from insect sequences NP_476592.1, NP_724162.1, XP_319838.3, EDS39158.1, EAT37246.1, and EAT37247.1. Duplicate sequences, sequences containing X as an amino acid, and sequences over 700 amino acids in length were all removed. The resulting sequences were aligned and a phylogenetic tree was generated using a split value of 0.12. After removing partial sequences, clusters were identified from a sequence similarity table generated by MOE.B. mori DHPAAS native sequence was synthesized by GeneArt (Invitrogen) and cloned into the pE-SUMO vector with kanamycin resistance (LifeSensors Inc.) via BsaI restriction sites. Mutations were generated using overlapping PCR with primers shown in Supplementary Table E. coli grown aerobically in LB medium. After induction, culture temperature was lowered to 14–16 °C. After overnight incubation, cells were pelleted by centrifugation, resuspended in phosphate-buffered saline (PBS) and lysed by sonication while being cooled over ice. After centrifugation, clarified lysates were loaded onto HiTrap TALON or HisTrap HP columns (GE Life Sciences). After loading, columns were washed with PBS and 10–20 mM imidazole. Recombinant DHPAAS was eluted with 450–1000 mM imidazole. Buffer was changed to PBS supplemented with PLP using Amicon Ultra-15 centrifugal filters (Millipore).The full length 2O at a ratio of 7:2:1 was used as the mobile phase. DHPAAS reaction components were analyzed under UV light before ninhydrin staining.TLC was performed on aluminum plates coated with silica gel 60 F254 (Merck Millipore). A mixture of 1-butanol, acetic acid and H36. Five concentrations of fresh L-DOPA (TCI), dopamine (TCI), DHPAA (Santa Cruz Biotechnology), THP hydrobromide (Sigma) and reticuline were analyzed to generate quantitative standard curves. Quantifier MRM transitions of 198.10 > 152.10(+), 154.10 > 91.05(+), 151.30 > 123.15(−), and 288.05 > 164.15(+) were used for L-DOPA, dopamine, DHPAA and THP, respectively. Qualifier MRM transitions of 154.10 > 137.05(+), 151.30 > 122.10(−), and 288.05 > 123.15( + ) were used for dopamine, DHPAA and THP, respectively. MRM transitions of 330.10 > 192.00( + ), 330.10 > 137.15( + ) and 330.10 > 177.20(+) were used for reticuline. Separation was performed at 0.25 mL/min on a Discovery HS F5-3 column  heated to 40 °C, with a mobile phase gradient of 0.1% formic acid in water to 0.1% formic acid in acetonitrile. Chiral analysis of -THP was performed using the Shimadzu LCMS-8050 system operated in MRM mode, together with an Astec CYCLOBOND I 2000 column  heated to 35 °C and a mobile phase gradient of 90% acetonitrile to 50 mM NH4OAc (pH 4.5) flowing at 0.3 mL/min.Substrate and products were identified with Shimadzu LCMS-8050 and LCMS-8060 ESI triple quadrupole mass spectrometers. Quantitative analysis was performed using the Shimadzu LCMS systems operated in multiple reaction monitoring (MRM) mode with the following parameters: ionization, ESI; DL temp., 250 °C; block heater temp., 400 °C; interface temp., 300 °C; nebulizing gas flow, 3.0 L/min; drying gas flow, 10.0 L/min; heating gas flow, 10.0 L/min. The ion source was connected to a Shimadzu Nexera X2 UHPLC systemL-DOPA to a final volume of 40 µL. A final concentration of 1.875 mM L-DOPA was used together with 2.5 mM sodium ascorbate (Wako). Reactions were started at room temperature (23–24 °C) and transferred to 4 °C after 8 h. At various time points, 2 µL of each reaction was diluted into 98 uL methanol supplemented with ascorbate and camphor sulfonic acid standard. Dilutions were performed in triplicate. DHPAAS reaction dilutions were immediately stored at −30 °C until triplicate LC-MS analysis. In vitro data were analyzed with Prism 7.For quantification of aromatic DHPAAS substrates and products, soluble DHPAAS (2–3 µg) in PBS was mixed with an aqueous solution of 2O2 production was analyzed in 96 well plates using a fluorometric hydrogen peroxide assay kit (Sigma). 0.6–0.8 µg soluble DHPAAS in PBS (20 µL) was mixed with varying concentrations of L-DOPA (10 µL), followed by the addition of 30 µL peroxidase enzyme and fluorescent substrate mix (Sigma). Fluorescence was detected with a SpectraMax Paradigm microplate reader (Molecular Devices). Kinetic data included eight independent conditions for wild-type DHPAAS (one outlier), Asn192His variant (two outliers), and Phe79Tyr-Tyr80Phe variant, while 15 independent conditions were used for the Phe79Tyr-Tyr80Phe-Asn192His variant. Kinetic data was analyzed using the kcat function of Prism 7 with outlier elimination.HDHPAAS sequences were PCR amplified with primers containing NcoI and XhoI restriction sites  was then added to each culture, followed by addition of PLP to a final concentration of 4.86 µM. Culture temperature was decreased to 16 °C 12.9 h. after the addition of L-DOPA. Culture samples (300–500 µL) for four time points were filtered through Amicon Ultra 0.5 mL centrifugal filters (Millipore) with a molecular weight cut-off of 3000 Da. Approximately 4–5 mg ascorbate was added to each culture 22.7 h. after substrate addition, and cultures were moved to 4 °C. Finally, 49.8 h. after substrate addition, cultures were centrifuged at 4500 × g, followed by collection and analysis of supernatants. Culture samples were diluted in methanol with camphor sulfonic acid and ascorbate for quantification of dopamine, DHPAA and THP. Individual dilutions were analyzed three times for each condition, and in vivo figures with error bars were generated using Prism 7.DHPAAS was transformed, together with a pACYC184 derived vector containing C. japonica 4-OMT, CNMT, and 6-OMT, into BL21(DE3). Resulting E. coli reticuline bioproducers were selected with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol. Reticuline production was tested in M9 minimal medium supplemented with 2% glucose. Cells were grown to an OD600 of 0.2–0.3 before the addition of 0.5 mM IPTG, 450 µM L-DOPA, and 4.54 mM sodium ascorbate. 17.2 h. after substrate addition PLP was added to 54.8 µM along with a second addition of an additional 444 µM ascorbate. SAM was added 23.5 h after substrate addition to a final concentration of 9.7 µM. Reticuline was produced at 25 °C with shaking. Filtered culture medium was diluted in acetonitrile with camphor sulfonic acid and ascorbate for quantification of dopamine, DHPAA, THP, and reticuline. Single cultures of Phe79Tyr-Tyr80Phe and Phe79Tyr-Tyr80Phe-Asn192His DHPAAS containing E. coli were tested. Duplicate cultures of wild-type and Asn192His DHPAAS containing E. coli were tested. Each dilution was measured in duplicate, resulting in duplicate measurements for Phe79Tyr-Tyr80Phe and Phe79Tyr-Tyr80Phe-Asn192His DHPAAS-mediated reticuline production, and quadruplicate measurements for wild-type and Asn192His DHPAAS-mediated reticuline production. In vivo figures with error bars were generated using Prism 7.pTrcHis2B containing 36.For optimization of reticuline titers, 3HC and 3HNMC  were analyzed together with DHPAA, THP, reticuline, and 115 central metabolites on the Shimadzu LCMS-8050DHPAAS in pTrcHis2B, DHPAAS-Phe79Tyr-Tyr80Phe-Asn192His in pE-SUMO, and C. japonica 4-OMT, CNMT and 6-OMT in pACYC184. For the first step of THP production, this three plasmid system was initially grown at 37 °C in TB with no glycerol supplemented with 1.5% glucose, 100 µg/mL ampicillin, and 50 µg/mL kanamycin. After reaching OD600 0.38, IPTG was added to a final concentration of 0.5 mM IPTG. 1.5 h after induction, the temperature was lowered to 25 °C. At 5.5 h post-induction, cells were collected by centrifugation at 4000 g, stored overnight at −80 °C, and the pellet from ~43 mL culture was resuspended in M9 with reduced calcium, 0.2% Triton X-100, 1.5% glucose, 10 μM PLP, 10 mM sodium ascorbate, and 1 mM L-DOPA, to a final volume of 6.5 mL. After mixing, cultures were kept at 24–25 °C for 1.5 h and then centrifuged at 5000 × g to assist condensation of DHPAA with dopamine in the supernatant. Twenty-five hours after substrate addition, the static culture was centrifuged again at 5000 × g and the THP containing supernatant was used for the next downstream BIA production step.BL21(DE3) was transformed with wild-type 4-OMT from C. japonica together with 6-OMT and CNMT from P. somniferum was transformed into BL21(DE3). This second BIA producer was also initially grown at 37 °C in TB with no glycerol supplemented with 1.5% Glucose and 100 µg/mL ampicillin. After reaching OD600 0.78, IPTG was added to a final concentration of 0.5 mM. 1.5 h after induction, the temperature was lowered to 25 °C. 5.5 h post-induction, cells were collected by centrifugation at 4000 × g, stored for 2 nights at −80 °C, and the pellet from ~46 mL culture was resuspended in the supernatant from the first step. Downstream BIA production was then tested at 25 °C with shaking. Diluted samples of culture medium were analyzed with three independent measurements for each condition.For the second step of increased downstream BIA production, pET23a containing Further information on experimental design is available in the Supplementary InformationPeer ReviewReporting SummarySource Data


The risk of transmission of infectious diseases from allograft to recipient is well known. Viruses and bacteria are the most frequent causes of transmissible infections. Donor-derived invasive aspergillosis is rare and occurred under particular circumstances. We report 2 cases of kidney transplant recipients who acquired aspergillosis from a single donor. The risk of transmission of infectious diseases from allograft to recipient is well known, although nowadays uncommon Cases of donor-derived invasive aspergillosis (IA) in transplant patients are unusual 22.1C. difficile infection. On day 7 after admission, he presented with fever and an acute neurologic event requiring ICU admission and intubation. A CT scan demonstrated bilateral intraparenchymal hematomas with uncal herniation and new bilateral lung infiltrates.The grafts came from a 50-year-old man with a history of alcoholic cirrhosis, several episodes of spontaneous bacterial peritonitis requiring antimicrobial treatment, and a recent admission due to acute alcoholic hepatitis that was treated with high-dose corticosteroids. Fifteen days after discharge he was readmitted with acute liver failure that again required corticosteroids and a relapse of Klebsiella pneumoniae that was treated with meropenem. The patient died on his fifth day at ICU. Death was attributed to a cerebral hemorrhage resulting from severe liver failure with massive bronchoaspiration.Bronchoalveolar lavage (BAL) culture revealed extended-spectrum beta-lactamase producing (ESBL) His potential as a kidney donor was based on a renal ultrasound that showed a simple cyst in the cortex of the left kidney (1.3 cm) and a Doppler ultrasound image that revealed adequate vascular flow in both kidneys. Accordingly, the transplant committee accepted his kidneys for transplant. His liver and heart were not used as grafts.2.2He was a 56-year-old man who had received a liver transplant (LTx) 15 years earlier, for which he took cyclosporine A (CyA) and mycophenolate mofetil (MMF). His general progress was good, and his graft function adequate. Three years after the LTx he experienced hepatitis C relapse and was treated with interferon and ribavirin. Since then, the patient has maintained good liver function.He developed end-stage renal disease (ESRD) due to hepatitis C associated–membranoproliferative glomerulonephritis. Residual diuresis was 100 mL/24 h, and he had been receiving hemodialysis for the past 36 months. He received a kidney transplant from a deceased donor with 3 HLA mismatches. The pretransplant biopsy graft contained 32 glomeruli, with no involvement of the vascular or tubulointerstitial compartment.The surgical procedure was uneventful, and the immunosuppression regimen included basiliximab, methylprednisolone, CyA, and MMF. Cold ischemia time was 6 h, and, despite good graft perfusion, he had delayed graft function and required hemodialysis on day 2 after transplantation.K. pneumoniae was identified in a BAL culture.Given the donor's history of respiratory infection, empirical ceftriaxone was started from day 0 to day 3 after surgery and was then changed to meropenem as soon as ESBL 2.3He was a 56-year-old man with ESRD secondary to nephroangiosclerosis. He had been on hemodialysis for 3 years. Residual diuresis was 1500 mL/24 h, and he underwent deceased donor kidney transplantation with 3 HLA mismatches. The pretransplant biopsy graft contained 39 glomeruli, with no involvement of the vascular or tubulointerstitial compartment.His immunosuppressive treatment included basiliximab, methylprednisolone, tacrolimus, and MMF. The surgical procedure was uneventful, and cold ischemia time was 10 h. He had delayed graft function despite good graft perfusion. He also received empiric ceftriaxone from day 0–3 post-transplant, and was then switched to meropenem.2.4At the time of kidney explantation and transplantation, the urologist noticed that one of the kidneys had a cyst. However, kidney biopsy specimens stained using rapid techniques in the pathology and microbiology departments were immediately reported as “normal”, and transplantation was performed.Aspergillus fumigatus. With this information, analysis of the previous renal biopsies reassessed with specific fungal stains  revealed filamentous fungi, which were subsequently confirmed by the histopathologist.On day 1 after surgery, a filamentous fungus started to grow in the culture plate of the donors’ respiratory sample (BAL). It was later identified as Despite the fact that both recipients were in a stable clinical situation and given the potential presence of IA, elective nephrectomy was performed on day + 3 after transplant in both cases. Surgery was uneventful, and the graft and vascular sutures were resected. In patient 1, the gross appearance of the kidney and the transverse section was normal. Samples of the cortex, medulla, hilum, and vascular sutures were sent for culture and histology.In patient 2, the kidney presented several parenchyma cavitations. Samples of the same areas were sent for culture and histology.A. fumigatus was isolated in samples from the cortex and vascular sutures. Tissue invasion was confirmed, and real-time PCR was positive for Aspergillus spp. in both cases. Chest radiographs were normal in both cases after graft extraction. Serological and urinary detection of In both cases, A. fumigatus was genotyped directly from positive PCR samples  using a highly discriminatory microsatellite procedure Short Tandem Repeats Aspergillus fumigatus (STRAf). Clinical isolates were not stored and consequently were not genotyped. All genotypes found were identical and showed the same allele composition for the 9 markers.Antifungal treatment was initiated in both cases before extraction. In patient 1, liposomal-amphotericin B (3 mg/kg/day) was preferred in order to minimize drug interactions with azole derivatives and the immunosuppressive therapy he received for the LTx. Voriconazole (6 mg/kg/12 h) was chosen for patient 2 on day 1, followed by 4 mg/kg/12 h, with weekly therapeutic drug monitoring and subsequent dosage adjustment if needed.Both patients were followed for 1 year and remain asymptomatic.We report 2 cases from the same donor in which the early diagnostic and therapeutic approach prevented potentially fatal IA.3Our episodes exemplify the potential risk of IA transmission to recipients from donors with risk factors for invasive fungal infections. The donor we report had received broad-spectrum antibiotics and corticosteroids and developed IA affecting the lung, CNS, and kidneys. There are 2 major types of donor-derived infections: those that are easily detected by donor and recipient screening  and those not easily detected despite routine donor screening Candida is the most common cause and is not always clearly related to the donor Donor-related fungal infections are uncommon and reported as small case series or case reports Transmission of filamentous fungi through organ donation, although infrequent, occurs under specific clinical circumstances Most donor-derived filamentous fungal infections occurred in kidney transplant recipients (91%) A. fumigatus (>100cfu/mL) was reported 24 h after donor death in the BAL sample. There was no previous calcofluor white stain. These samples are not routinely processed, unless at the clinician's request. As with most cases reported in literature, the present cases underwent graft explantation. It is important to mention that calcofluor white stain is mandatory in potential donors with a high risk of IA.In the cases we report, the growth of As these cases demonstrate, aggressive diagnostic efforts must be undertaken if there is suspicion of donor-derived fungal graft infection. Percutaneous graft biopsy for histopathology and fungal culture, graft imaging, and early surgical exploration are all part of the diagnostic approach. Our patients also had positive BDG titers in serum and urine, and this can purportedly be used in future episodes as early diagnostic tools.Donor-related IA is associated with severe consequences, such as graft loss (83%) and a high mortality rate (17%) Invasive fungal infections represent a diagnostic and therapeutic challenge. However, awareness of the risk of donor-derived fungal infections may help clinicians to properly identify donors potentially capable of transmitting them and facilitate timely recognition of transmission to recipients.


In August 2018, CDC noted an increased number of reports of patients having symptoms clinically compatible with acute flaccid myelitis (AFM), a rare condition characterized by rapid onset of flaccid weakness in one or more limbs and spinal cord gray matter lesions, compared with August 2017. Since 2014, CDC has conducted surveillance for AFM using a standardized case definition (3]), or “not a case.”Patients who meet the clinical case criteria for AFM, defined as acute flaccid limb weakness, are classified using the Council of State and Territorial Epidemiologists case definitions of “confirmed” , “probable”  , 6 as pr3 ; most had a lymphocyte predominance. Median CSF protein and glucose were 47 mg per dL  and 59 mg per dL , respectively. The median interval from limb weakness to CSF collection was 1 day . The median interval from sign or symptom onset to CSF collection was 7 days  for respiratory illness, 4 days  for gastrointestinal symptoms, and 3 days  for fever.Among 78 (98%) confirmed cases with available CSF results, 65 (83%) had pleocytosis, with a median cell count of 103 cells per mmCDC conducts enterovirus/rhinovirus (EV/RV) testing for all patients meeting the clinical criteria for AFM, when specimens are available. Of the 80 confirmed cases in 2018, testing was performed on a total of 125 clinical specimens from 71 (89%) patients, including 21 CSF, 59 upper respiratory, and 45 stool/rectal swab specimens . Among thttps://www.cdc.gov/acute-flaccid-myelitis/afm-surveillance.html), CDC did not receive reports of large outbreaks of severe respiratory illness in 2016. Further, there has been limited detection of pathogens in CSF in these cases; virus identified in CSF would be considered etiologic. Almost all patients with AFM have reported signs and symptoms consistent with viral illness in the weeks preceding limb weakness. Clinical, laboratory, and epidemiologic evidence to date suggest a viral association. CDC and collaborators continue to investigate risk factors for AFM and to study the causes and mechanisms of AFM.Because some enteroviruses can cause acute flaccid limb weakness, and there was a temporal association with AFM and a nationwide severe respiratory outbreak of EV-D68 in 2014 (https://www.cdc.gov/acute-flaccid-myelitis/hcp/index.html. Patients with acute flaccid limb weakness should be reported to their health departments as soon as possible regardless of laboratory or MRI findings.Parents and caregivers are urged to seek immediate medical care for a child who develops sudden weakness of the arms or legs. In the evaluation of a child with acute flaccid limb weakness, clinicians are advised to inquire about recent fever with or without antecedent respiratory or gastrointestinal symptoms and to collect timely specimens for viral testing, including CSF, serum, respiratory, and stool specimens. Additional information for clinicians is available at


Mesenchymal stem cells (MSCs) have emerged as one of the promising treatment options for Alzheimer’s disease (AD). Although many studies have investigated on the efficacy of MSCs in AD, how MSCs actually change following exposure to the AD environment has not been studied extensively. In this study, we investigated on the potential of AD patient-cerebrospinal fluid (CSF) samples to be used as a formulation of MSCs and its application in AD therapeutics. When Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) were stored in the CSF of AD patients, the stemness of WJ-MSCs was preserved. Furthermore, several genes were upregulated following storage in AD CSF. This signified the therapeutic potential of CSF formulation for AD therapy. Overall, these findings suggest that CSF from AD patients can be an optimal source for MSC formulation. Alzheimer’s disease (AD) is the most common neurodegenerative disease for which no disease modifying therapy exists so far5. MSCs have been considered as one of the treatment options for AD since these cells have shown a variety of effects such as reduction of beta amyloid (Aβ) levels, modulation of neuroinflammation, enhancement of endogenous neurogenesis, and also improvement in the behavioral performance of AD-transgenic mice13.Originally found in the stroma of bone marrow16. Several preclinical studies have looked into which routes are more feasible to adminster MSCs in AD patients. Animal studies from our group, for instance, showed that intracerebroventricular17 or parenchymal injections of MSCs8 might be more effective than intravenous18 or intra-arterial injections19.A growing body of clinical studies is underway to investigate the safety and efficacy of MSCs when administered to AD patients20. From the isolation of cells to the preparation of the final stem cell therapeutics product, a critical point to consider is whether unintended ingredients could be included21. Current formulations for MSCs often include supplements to promote the survival of MSCs. However, some supplements may not exist in the body, thus possibilities of undesirable interactions between excipients and internal factors of recipients following injection cannot be ruled out. Therefore, it is imperative to produce an optimized form of the formulation where the excipients have been well estabilished chemically.While safety, efficacy, and route of administration have been addressed, the clinical grade production of MSCs as a drug product has not been investigated in-depth22. To perform repeated intracerebroventricular injections, insertion of a device such as an Ommaya reservoir has to be preceded prior to the first stem cell injection, which makes the collection of CSF from AD patients easier. MSC formulation using the CSF from the patient to whom the MSCs will be injected into may therefore ensure the safety of the therapeutic agent. Second, since CSF flow is part of the Aβ clearance mechanism in AD24, CSF from AD patients might represent a microenvironment for AD. Therefore, MSCs that have been exposed to the AD patient CSF as a formulation would be preconditioned in the AD microenvironment prior to administration. This pre-exposure of MSCs to the AD microenvironment might allow MSCs to better cope with the disease environment than naïve MSCs.When producing clinical grade MSCs, cerebrospinal fluid (CSF) samples from AD patients may have the potential to be applied as an optimal formulation of MSCs for the following reasons. First, one of the most feasible routes of administration for MSCs is the intracerebroventricular route, where cells have higher chances to penetrate into the parenchyma of the brainIn the present study, we stored human Wharton’s jelly-derived MSCs (WJ-MSCs) in CSF samples obtained from four different AD patients and three normal controls under hypothermic conditions (4 °C). We then investigated whether the viability and stemness of WJ-MSCs were compromised following exposure to CSF of AD patients or normal controls. In addition, we explored changes in gene expression levels of WJ-MSCs to assess the therapeutic effects of AD CSF formulation.42 level, marked as * in Table 42 ratios : female (F) = 2:2, age range 50–60] who underwent 18F-florbetaben amyloid positron emission tomography (PET) scans were amyloid positive. Negative results were obtained from the three cognitively normal controls  who also underwent the same amyloid PET scans. Although some samples showed unexpected levels of CSF biomarkers  analysis after Annexin V/7-AAD staining Fig. . Images 26. Like WJ-MSCs stored in MEMα 1x, WJ-MSCs stored in normal and AD CSF expressed the following cell surface markers: CD90, CD73, CD105, CD166 and also did not express the following hematopoietic markers: CD14, CD11b, HLA-DR, CD34, CD45, and CD19 D19 Fig. . Such reExpression levels of mRNA for MEMα 1x, normal CSF, and AD CSF groups were analyzed by using the Human Mesenchymal Stem Cell RT² Profiler PCR Array. The expression patterns of WJ-MSCs exposed to AD CSF and normal CSF were compared to those of WJ-MSCs exposed to MEMα 1x. Based on the PCR array data, scatter plots and heatmaps were analyzed Fig. . The mRNGene expression clustering for upregulated and downregulated genes was performed using the MeV software Fig. . The funThe 10 most enriched annotation clusters of significantly changed genes included items related to angiogenesis, signal peptide, negative regulation of programmed cell death, regulation of cell proliferation, extracellular region, tube development, morphogenesis of branching structure, cellular component morphogenesis, regulation of neurogenesis, and sensory organ development.27. Previous studies also showed that proteins secreted from MSCs induced clearance of Aβ proteins9, promoted neurogenesis and also synaptogenesis17. These studies indicated that a therapeutic interaction existed between the paracrine factors secreted by the MSCs and the endogenous progenitors present in the brain. While former studies were mainly focused on the therapeutic effects of MSCs, the cellular status of MSCs exposed to the AD brain microenvironment has not been fully elucidated.Recent studies have reported on the neuroprotective and neurotrophic features of MSCs29 and regulate quiescence and activation of stem cells in the brain30. Other studies have used CSF to transdifferentiate MSCs into neural cells. For example, MSCs cultured in-vitro in CSF (as a substitute of culture media) transdifferentiated into neural-like cells32. However, these studies have focused on elucidating the effects of CSF on stem cells under normal and not disease conditions.Several studies have examined the effects of CSF on stem cells. Growth factors found in the CSF have been reported to affect stem cell proliferationIn this study, we applied CSF samples from AD patients as a formulation of WJ-MSCs. For comparison, MEMα 1x, which is conventionally used for MSC formulation was included, and normal control CSF samples were also included to determine whether AD pathology can possibly alter the therapeutic effects of MSCs. According to the results obtained from this study, the viability and stemness of WJ-MSCs were both preserved after exposure to AD CSF under hypothermic conditions. These results confirmed the safety of AD CSF to be used as a potential source of formulation.We further explored changes in gene expression levels of WJ-MSCs exposed to AD and normal CSF samples. Based on analysis of functional annotation clustering, WJ-MSCs stored in AD CSF expressed genes related to enhancement of extracellular transport and signal peptide, which indicates an increase in paracrine activity. These genes are known to exhibit neuroprotective and neurotrophic features such as negative regulation of apoptosis, regulation of cell proliferation, and regulation of neurogenesis. Furthermore, an increase in the expression of genes involved in cell migration or cell adhesion was also observed, which indicates potential beneficial effects on cell survival following administration. These results suggest that AD CSF may act as an optimal formulation for MSCs that will be injected back to the AD patient from whom the CSF sample was obtained from Fig. .Figure 6Our study has several limitations. First, our study was based on a small number of patients. Therefore, further research involving a large number of patients is warranted. Second, although we measured the gene expressions of MSCs to investigate functional changes, we have not yet assessed the efficacy of MSCs in animal disease models . Nevertheless, to the best of our knowledge, this is the first study which has investigated the fate of MSCs in AD CSF, the interaction between MSCs and CSF samples, and the potential of AD CSF as a formulation source. Our approach may have several advantages. First, patients will be reinfused with their own CSF, which may contribute to minimizing the side effects of allogeneic MSC administration. Autologous MSCs are also available but not as cost-effective due to the requirement of large scale cell expansion, compared to allogenic MSCs. Moreover, the surgical process involved in isolating MSCs is difficult to perform in AD patients due to their age. Second, to achieve repeated injections of MSCs into the lateral ventricle of patients, a device such as an Ommaya reservoir has to be surgically implanted. Therefore, collection of CSF from the patient may not be as invasive as expected. In order to ensure patient safety, CSF collection must be performed under closed, sterile procedures. CSF collected under sterile conditions can be used as an optimal formulation of MSCs produced from the GMP facility. Third, using AD CSF as a formulation of MSCs allows MSCs to pre-adapt to the disease environment and to also become pre-activated prior to administration. The use of CSF from AD patients as a source of formulation may enhance the overall efficacy of AD MSC therapy. This approach can also be applied to a wide range of neurological diseases.33 for probable AD and also had positive 18F-florbetaben PET scans35. Patients with neurological diseases other than AD were excluded from the study. The three normal controls were recruited from orthopedic clinics in our hospital who underwent spinal anesthesia to receive knee surgeries. All of them met the criteria for normal elderly36, had normal Mini-mental State Examination scores defined by age/sex matched cohort37, and also had negative 18F-florbetaben PET scans.This study was approved by the Institutional Review Board of Samsung Medical Center . In accordance with the guidelines approved by the Institutional Review Board (IRB) of Samsung Medical Center, CSF samples were collected with informed consent from four AD patients and three normal controls. All AD patients fulfilled the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria1–42, P-tau181P , and T-tau concentrations were examined from the CSF samples by using the IINOTEST (ELISA) assay. Then the ratios of tau (both t-tau and p-tau) to Aβ42 were obtained to validate the difference between normal controls and patients, as these ratios are known to be more accurate than the level of Aβ42 and tau38.A total of 10–12 mL of CSF was obtained from each of the four AD patients and 3 mL from the three normal controls by lumbar puncture . Within two hours from collection, CSF samples were centrifuged at 4,000 g for 10 minutes at 4 °C. AβAccording to the Institutional Review Board of Samsung Medical Center, after written informed consent was received by all of the patients, umbilical cord tissue was obtained from healthy patients undergoing childbirth. Wharton’s jelly mesenchymal stem cells (WJ-MSCs) were isolated and cultivated according to the standard operation procedures (SOPs) of the Good Manufacture Practice (GMP) facility at Samsung Medical Center.6 cells/1.5 mL, which corresponded to the final product vial concentration used in the GMP facility. The cells were stored in four different AD CSF samples at 4  C for 72 hours. For the control group, WJ-MSCs were suspended in serum and phenol red free MEMα 1x  and three normal CSF samples.Passage 5 WJ-MSCs suspended in CSF samples were stored under hypothermic conditions at a concentration of 5.5 × 10Cell proliferation and viability were assessed by using the CCK-8 Cell Counting kit  and Annexin V apoptosis detection kit with 7-AAD . MSCs stored for 72 hours under hypothermic conditions from the eight different samples (including the controls) were harvested and analyzed. To determine the number of viable cells in the stocked vials, the cells were stained with Annexin V and 7-AAD antibodies according to the manufacturer’s instructions. The cells were resuspended in 500 μL of 1x staining buffer. Then, 5 μL of Annexin V and 7-AAD antibody were added to the binding buffer for 20–30 minutes at room temperature in the dark, prior to being analyzed by flow cytometry .3/well), and after 24 hours, CCK8 assay was performed. CCK-8 solution (10 uL) was added to each well, followed by incubation for 1 hour at 37 °C. The absorbance of CCK-8 was measured at 450 nm by using a microplate reader .To analyze the viability of the cells at various time points , MSCs stored in CSF were seeded onto 96-well plates , CD34, CD45 and CD19 . At least 10,000 events were acquired by using the BD FACS Verse flow cytometer, and the results were analyzed by using the BD FACSuite software version 10. Flow cytometry for appropriate isotype controls were also performed.2 and expanded until cells reached 80–90% confluency. For osteogenic and adipogenic differentiation and the respective immunostaining experiments, cells were incubated in differentiation media according to manufacturer’s instructions . Differentiation medium was replaced every 3 days. After 2 weeks, differentiated cells were stained using the following staining methods: osteogenic; Alizarin Red S, adipogenic; Oil Red O. Osteocytes were fixed with 4% paraformaldehyde (PFA) for one hour and then washed with PBS not including both calcium and magnesium . Mineralization of the extracellular matrix was visualized by staining with 40 mM Alizarin Red S , pH 4.2, for 5 minutes. Adipocytes were fixed with 4% PFA, washed in 60% isopropanol, and subsequently incubated for 10 minutes with Oil-Red O  to visualize the lipid droplets. Cells were then washed in isopropanol and counterstained with hematoxylin.Another way to test stemness is to examine the differentiation capabilities of MSCs. WJ-MSCs stored under hypothermic conditions for 72 hours were seeded onto 6-well culture plates at a density of 5000 cells/cm5 cells were pelleted in 15 mL conical tubes. Subsequently, the cell pellet were suspended in 500 μL of chondrogenic differentiation medium containing high-glucose Dulbecco Modified Eagle Medium  supplemented with 500 ng/mL of bone morphogenic protein-6 (BMP-6) , 10 ng/mL of recombinant human transforming growth factor-β3 , 1% ITS , 50 μg/mL of ascorbic acid-2-phosphate, 0.6 μg/mL of dexamethasone, 40 μg/mL of L-proline, and 100 μg/mL of sodium pyruvate. Differentiation medium was replaced every 3 days. Chondrocytes were fixed with 4% PFA, dehydrated by using ethanol, and embedded in OCT compound . Blocks were sectioned and the sections were stained with safranin-O .For chondrogenic differentiation, 2 × 1041.Previously described methods were used to quantify the differentiation potential between each samples6 cells by using Trizol , according to the manufacturer’s instructions. Integrity of isolated RNA was evaluated using the RT2 RNA QC PCR Arrays (Qiagen) and cDNA was synthesized from 500 ng of the total RNA using the RT2 First Strand Kit . The samples were analyzed using the RT2 Profiler PCR Array. The reaction mix was prepared from 2x RT2 qPCR Master Mix and 102 μL of sample cDNA. 10 μL of this mixture was added into each well of the PCR Array. Altogether, 84 different genes were simultaneously amplified in the sample. A melting curve analysis was performed to verify that the product consisted of a single amplicon. PCR arrays were performed in 96-well plates by using a QuantStudio 6 flex real-time PCR system . Data were analyzed via the QuantStudio software and the Ct values were determined for each gene. The thresholds and baselines were set according to the manufacturer’s instructions . The fold change in gene expression (compared to the positive control: WJ-MSCs) was calculated using the ΔΔCt method. Compared to the control (MEMα 1x), gene expressions with a fold change ≥2 was only considered. Clustering analysis of altered gene expressions was performed by using the MeV software (Ver. 4.9.0).To determine if there is a significant difference in genetic expression of MSCs following hypothermic storage in AD CSF, gene expression analysis was performed. The Human Mesenchymal Stem Cell RT² Profiler PCR Array  was used to evaluate the expression of 84 specific genes related to MSCs. After 72 hours of hypothermic storage, the total RNA was isolated from 1 × 1043). For GO (Gene Ontology) Term analysis, we studied the Cellular Component (CC), Molecular Function (MF) and Biological Process (BP) categories using the GO FAT default settings. For functional annotation searches, we set the following parameters: CC, threshold count 2, EASE 0.01, Benjamini 0.05 (resulting in 15 chart records); MF, threshold count 2, EASE 0.01, Benjamini 0.05 (resulting in 9 chart records); BP, threshold count 10, EASE 0.01, Benjamini 0.05 (resulting in 60 chart records); for functional annotation clusters, medium stringency (resulting in 52 clusters). Enrichment values (GO Terms), enrichment scores (annotation clusters), and statistical determinants  were calculated by the DAVID software.Functional analysis was conducted to examine alterations in gene expression following storage of MSCs in AD CSF. For annotation analysis, official gene symbols were uploaded into the DAVID  informatics tool (DAVID Bioinformatics Resources 6.7The results are an average of three independent experiments. Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons of each samples between groups were performed using a one-way ANOVA test (both between groups and within groups). Differences were considered statistically significant when P < 0.05. All the statistical analyses were performed using SigmaPlot, version 12.5 and SPSS software, ver 19.0 for Windows.Supplementary Information