In the present work, modified embedded atom potential and large-scale molecular dynamics’ simulations were used to explore the effect of grain boundary (GB) segregated foreign interstitials on the deformation behavior of nanocrystalline (nc) iron. As a case study, carbon and nitrogen (about 2.5 at.%) were added to (nc) iron. The tensile test results showed that, at the onset of plasticity, grain boundary sliding mediated was dominated, whereas both dislocations and twinning were prevailing deformation mechanisms at high strain. Adding C/N into GBs reduces the free excess volume and consequently increases resistance to GB sliding. In agreement with experiments, the flow stress increased due to the presence of carbon or nitrogen and carbon had the stronger impact. Additionally, the simulation results revealed that GB reduction and suppressing GBs’ dislocation were the primary cause for GB strengthening. Moreover, we also found that the stress required for both intragranular dislocation and twinning nucleation were strongly dependent on the solute type. In literature, several experimental and theoretical studies on mechanical properties of nc metals reveal that strengthening could be achieved not only by controlling their grain size but also by tailoring and doping their grain boundary ,4,5,6,7.nitrogen ,18,19. Initrogen  demonstrnitrogen  reveal tnitrogen .In other work by Takuro et al. , they stDespite extensive experiments that strongly indicate that carbon and nitrogen segregation to GBs can improve the strength of nc ferrite, the roles of carbon and nitrogen in the strengthening and plasticity of these materials are still not fully understood. In the present work we utilized the large-scale atomistic simulations with classical interatomic potentials for Fe-C and Fe-N to investigate the effect of carbon and nitrogen on the deformation mechanisms of ferritic steel. Furthermore, the impact of carbon and nitrogen on the cohesive energy, atomic volume, and stacking faults was investigated. Such investigations will improve the knowledge on the mechanical properties of the nc ferritic steels and will contribute to widening the applications of these materials.D = 14.12 nm, was built by filling in a cubic box with randomly distributed seeds. Then bcc lattice grains with random orientations were generated from the seeds. The simulation box had size of 350 × 210 × 170 Å3 along the x, y, and z directions, respectively, containing about 1,100,000 atoms. To eliminate free surface effects, the simulation cell was periodic in all directions. In order to avoid high energy configuration, the overlapped atoms within 0.7 Å distances were removed, followed by structural minimization using the conjugate gradient method. Then, the system was thermally equilibrated to 300 K and 0 GPa pressure for 200 ps using isobaric/isothermal constant number of particles, constant pressure and constant temperature (NPT) ensemble, so that the total stress became zero. The time step of 1 fs was chosen for our simulations. The common neighbor analysis (CNA) [A nanocrystalline bcc iron sample, depicted in is (CNA)  was perfis (CNA) . In the 8 s−1, along x direction, whereas the pressure in both y and z directions was kept at 0. This strain rate was considerably high compared to the lab experiments; however, it was enough to capture qualitatively grain boundary activity as well as dislocations mediated by plastic deformation.During dynamic loading, the sample was subjected to uniaxial tensile load with constant strain rate, 5 × 10The interaction between Fe-C  and Fe-NDuring tensile simulation, the identification of dislocations was made via the dislocation extraction algorithm (DXA) , while tBefore applying the tensile deformation, the as-prepared samples underwent thermal annealing at 300 K for 200 ps. This procedure is important to allow the system to relax and remove undesired high-energy configurations. Here, we focused only on the role of the grain boundary segregation to the strength of nanocrystalline samples. Obviously, the GB area is characterized by atoms having higher energies compared to grain interior atoms due to the lack of ordering of GB atoms. In order to quantitatively assess the effect of the segregated atoms on the material strength, one may estimate the GB energy, given by ectively ,36. Howen bcc-Fe ,37.Additionally, the results of DXA analysis, as depicted in ’s model , in whicThe value of lable in ,19. It wIn the following we shall investigate the influence of solute elements on the plasticity of nc samples by analyzing the deformation mechanisms at different strain values. As mentioned in tion (1) .(1)ηvm=The spatial distribution of  of bcc-Fe single crystal with/without foreign atoms was studied see . The GPFutf) see . These vnd Bilby . Accordi<0.5 GPa ,44). The<0.5 GPa .On the other hand, we calculated the ratio, (Finally, the formation of vacancies during plastic deformation was observed. Obviously, as depicted in In the present study we performed MD simulations to explore the role of grain boundary segregated carbon and nitrogen on the plasticity of nanocrystalline bcc-iron. Our MD results indicate that GB strengthening occurred because of two primary mechanisms, the reduction of GB energy and the pinning of the GBs’ dislocations. Moreover, the flow stress of nc bcc-iron increased by about 15% and 8% upon addition of C or N, respectively. During tensile deformation, the grain boundary sliding was dominant at early stages of plasticity, while at large strain both dislocation activity and deformation twinning contributed to deformation mechanisms. Adding carbon or nitrogen to bcc-iron also reduced the free atomic volume fraction and, consequently, the grain boundary sliding resistance increased. Additionally, the calculated critical nucleation stress for twins and dislocation was higher for samples with solute atoms compared to pure nc bcc-iron. The simulation results related to the generalized planar fault energy curve clearly showed that the values of stacking and twinning fault energies varied considerably with carbon and nitrogen content.


Nicotiana tabacum L.) is a major cash crop in Yunnan, China, and the yield, chemical components, and their proportions decide the quality of tobacco leaves. To understand the effects of environmental factors (soil and climatic factors) on the yield and quality of flue-cured tobacco and determine the main regulating factors, we selected three flue-cured tobacco cultivars  grown in the Honghe Tobacco Zone. Indices related to yield and economic traits, chemical component properties, soil physical and chemical properties, and climatic factors at different planting sites, were evaluated. We used variance analysis, correlation analysis, and redundancy analysis (RDA) in this study. The results showed that the yield and chemical component properties of flue-cured tobacco, except for the number of left leaves and plant total sugar (PTS) content, were significantly correlated with climatic factors. Particularly, the yield increased in drier and sunnier weather. In terms of the carbon supply capacity, PTS, petroleum ether (PPE), and starch contents (PS) were higher under high-altitude and high-latitude climatic conditions, whereas for the nitrogen supply capacity, plant nitrogen (PTN) and nicotine (PN) contents improved under low-altitude and low-latitude climatic conditions. PTS, reducing sugar (PRS), potassium (PTK), chlorine (PCL), and PPE contents were negatively related to soil clay content, soil pH, and soil organic matter, whereas PRS and PTK contents were positively correlated with alkali-hydrolyzed nitrogen (AN). According to RDA, the soil clay, AN, available phosphorus (AP), and soil chlorine content (SCL) strongly affected the quality of flue-cured tobacco. The quality of the K326 and Yun87 cultivars was mostly influenced by moisture, whereas the quality of the Hongda cultivar was mostly affected by temperature. In conclusion, compared with soil properties, climatic factors more significantly affect the yield and quality of Honghe flue-cured tobacco leaves.Flue-cured tobacco ( Nicotiana tabacum L.) is a major cash crop in Yunnan, China. Yunnan produces approximately 750,000 t of flue-cured tobacco on 320,000 ha of land every year1 and approximately 50% of the total tobacco leaf yield in China2. Because of its unique climate and geographical environment, Yunnan Province produces high-quality flue-cured tobacco that has been applauded for its golden color, fragrant aroma, mild effect, and pure taste3. Tobacco can adapt to a wide range of conditions but is very sensitive to the environment. The quality of tobacco is related to various factors, including the climate5, crop rotation patterns7, soil properties9, and soil microbes11. The natural environment is the ecological basis for the development of high-quality tobacco leaves.Flue-cured tobacco  varieties to water stress differed at the proteomic level24. Moreover, climatic factors such as rainfall, temperature, and frost can affect the incidence and severity of disease in flue-cured tobacco4. To improve the potential of tobacco cultivars, it is necessary to understand the characteristics of cultivars under different environmental conditions.Ecological conditions have great impacts on the yield and quality of tobacco7, soil fertility25, and soil microbes26 in different tobacco-growing areas in China. For example, the organic matter and total nitrogen contents in soil should be suitable in regions of tobacco cultivation27. The lack of nutritional elements or imbalanced proportions will impede or cause abnormal changes in the growth and physiological metabolism of tobacco plants8. For example, high concentrations of total nitrogen induce excessive growth of tobacco28 and cause an imbalance of chemicals in the plants29, affecting the yield and quality of tobacco leaves. Moreover, the soil substrate is a determining factor for both aboveground and belowground biomass19. However, the combined effects of climatic factors and soil properties on the quality of tobacco leaves and their relative contributions remain unclear. In 2015, flue-cured tobacco in the Honghe Tobacco Zone was mainly planted in four counties (cities), Luxi (LX), Mile (ML), Jianshui (JS), and Shiping (SP) , Yunnan Province, China, an area belonging to the plateau subtropical monsoon climate region. The average annual rainfall is 1491 mm. The average annual sunshine is 1065–2300 h. In the middle of March, the temperature stabilizes, remaining above 13.0 ℃, then rises to 19.0–24.5 ℃ in April. The average temperature in May–August is above 20.0 ℃, and the average temperature in September is between 20.4 and 24.6 ℃, which is the most suitable temperature for flue-cured tobacco growth. The three main local cultivars  were used in the experiment and were planted in JS, LX, ML, and SP , crop rotation, and management of plant diseases and pest insects. These practices were recommended by the Tobacco Institute of Yunnan and are used in other Yunnan areasFloating breeding to obtain healthy and disease-free tobacco seedlings started from March 1 to 10.Transplanting under plastic film occurred from April 10 to 20. The transplanted seedlings were 5–7 cm in height with 4–5 leaves combined on one core. The transplant spacing for all cultivars was 120 cm × 50 cm, with a final density of 15,000–16,500 plants per ha.In 2015, irrigation methods included water cellars (41.67%), dammed ponds (19.00%), reservoirs (11.00%), rivers (11.00%), and mountain springs, water wells, and vauclusian springs (7.34%); 9.99% of the fields had no water conservancy facilities.The following are the main flue-cured tobacco rotation modes in the region: flue-cured tobacco → corn → flue-cured tobacco, flue-cured tobacco → wheat → flue-cured tobacco, flue-cured tobacco → rice → flue-cured tobacco, and flue-cured tobacco → others → flue-cured tobacco. In 2015, the flue-cured tobacco → corn → flue-cured tobacco mode was most frequently employed (75.33% of production).2O5:K2O ratios of 1:1:2.5–3.0 and 1:1:3.0–3.5, respectively. For the Hongda cultivar, pure nitrogen fertilizer was applied at a rate of 60–75 kg/ha with a N:P2O5:K2O ratio of 1:1:3.0–5.0.Pure nitrogen fertilizer was applied at a rate of 90–110 kg/ha for both the K326 and Yun87 cultivars, but with N:PProdenia litura (Fabr.), and Myzus persicae (Sulzer) have caused heavy losses in Honghe. Disease-control measures mainly include physical measures, chemical measures, and biological measures, such as crop rotation, disease-resistant cultivar selection, chemical pesticides, application of organic fertilizer, and deep turning (30 cm) and drying of field soil, while following the principle that biological control is the focus, physical control is auxiliary, and chemical control is supplementary.Black shank disease, root knot nematode disease, viral diseases, cutworms, To enable optimal growth and development of flue-cured tobacco, the lowest leaves (lugs) and the apical meristem must be removed, and the remaining effective leaves are known as “left leaves.” The numbers of left leaves per plant were 18–20, 16–18, and 18–20 for the K326, Hongda, and Yun87 cultivars, respectively. The squaring stage was around June 5 for K326 and Yun87, and around May 30 for Hongda; the height after removing the apical meristem was 100–110 cm for the K326 and Yun87 cultivars and 90–110 cm for the Hongda cultivar. The leaf samples were weighed to calculate yields, which were 10.67–12.01, 8.67–10.01, and 10.01–11.34 kg/ha for K326, Hongda, and Yun87, respectively. As flue-cured tobacco grows and matures, the fresh leaves turn yellow and mature layer-by-layer from the bottom of the plant. Two to three effective mature lower and middle leaves are harvested at a time, whereas four to six upper leaves are harvested at a time. The harvesting stage for the K326, Yun87, and Hongda cultivars was from June 30 to September 10. In our study, we collected the middle leaves in August after cultivating the plants for 85–95 days and topping for 30–40 days. At this time, the tobacco leaves were pale yellow, and 80% of the leaves had yellowed and were exhibiting white, bright main veins, white branch veins, downward rolled leaf apices and leaf margins, and marked mature plaques; mature leaves were collected on two to three occasions. After the tobacco leaves are picked, the leaf samples should be baked for a few days under different temperatures using an intelligent baking method.According to the National Standard (GB2635-1992), the grades of flue-cured tobacco were classified based on seven appearance grade factors, including maturity, leaf structure, identity, oil content, color, length, and residual injury, which were divided into 42 levels in total. For the sake of statistics, X, C, and B represent the lower, middle, and upper tobacco leaves, respectively; L and F represent the colors lemon and orange. Middle tobacco leaves of grade 3 with an orange color (C3F) were used for the analysis of chemical components in our study. The proportion of superior middle tobacco  and the proportion of superior tobacco (only superior grades) were estimated based on national purchasing data and data on the supervision and inspection of grade quality during the industry–commerce handover in that year. Additionally, each grade had an associated monetary value; this value was multiplied with crop yield to estimate the overall crop economic value .32. Gravel, roots, and large organic residues were manually removed before the soil samples were passed through a 2 mm sieve, then samples were stored in sealed bags in an ice cooler within 2 h of collection. In the laboratory, the samples were used to measure soil mechanical composition and soil properties . Tobacco plant samples of the three cultivars  were also collected from the soil sampling sites.We collected 39, 76, 87, and 42 soil samples from JS, LX, ML, and SP, respectively Fig. . The sam33. We calculated field temperatures, sunshine hours, rainfall, relative humidity from May to August. Sunshine hours and rainfall were calculated as sum values, and temperature and relative humidity were calculated as average values; the values for all parameters are summarized in Table The Honghe branch of the Yunnan Tobacco Company has access to 30 years 1980–2010) of meteorological data collected by the Yunnan meteorological observatory, including monthly temperature, rainfall, sunshine, and average relative humidity data. We also obtained monitoring data on temperature and precipitation as well as other meteorological data for 2013–2015. The data were standardized using an algorithm to obtain temperature, sunshine, rainfall, and humidity data for our analysis980–2010 C3F leaf samples were used to determine the final chemical compositions. The plant total nitrogen (PTN), potassium (PTK), and chlorine (PCL) were analyzed according to the YC/T161-2002, YC/T 173-2003, and YC/T 162-2011 methods, respectively. Plant total sugar (PTS) and reducing sugar (PRS) contents were analyzed according to the YC/T 159-2002 method. Plant nicotine (PN), starch (PS), and petroleum ether (PPE) contents were analyzed according to the LY/T1228-2015, NY/T1121.7-2014, and NY/T889-2004 methods, respectively. In this study, we also calculated the plant sugar/alkaloid (PSA) ratio and the total nitrogen to total alkaloids (PNA) ratio.Soil mechanical composition  was calculated according to the NY/T1121.3-2006 method, and soil pH according to the NY/T1377-2007 method. SOM, AN, AP, AK, and SCL were analyzed according to the NY/T1121.6-2006, LY/T1228-2015, NY/T1121.7-2014, NY/T889-2004, and NY/T1378-2007 methods, respectively.p < 0.05. Pearson correlation coefficients were used to describe the relationships between chemical composition of the tobacco leaves and soil and climatic factors. Furthermore, we carried out redundancy analysis (RDA) to determine the linkages between tobacco yield and quality and ecological factors19. The forward selection procedure in RDA, based on Monte Carlo permutation with 499 iterations, was performed to determine the most significant discriminating variables for tobacco yield and quality variables, and the significant variables (p < 0.05) are shown in the figures. Significance tests for RDA were performed using Canoco for Windows 4.5 . All statistical analyses were performed using SPSS version 17.0 .The normality and homogeneity of the residuals of the predicted values were checked in terms kurtosis and skewness, respectively. It was necessary to use rank cases to transform the yield, proportion, and SCL variables to meet parametric assumptions of normality and homogeneity. Significant differences among treatments were tested using analysis of variance (ANOVA) followed by Duncan’s test of significant differences at a significance level of Based on the yield and economic value per hectare, as well as the proportion of superior middle tobacco and abundance of left leaves of flue-cured tobacco, as shown in Table The variations in tobacco plant quality properties as influenced by cultivar type and planting site were analyzed by two-way ANOVA Table . The PN,Pearson correlation analysis was used to examine the relationships between the yield and internal chemical composition of flue-cured tobacco and soil properties and ecological factors Table . The resFurther RDA analysis showed that axes 1 and 2 explained 53.1% and 17.2% of the variation in the yield and quality of the K326 cultivar, respectively. Forward selection of the environmental factors in the RDA ordination showed that field rainfall and AN content explained 29% and 12% of the variation, respectively 39. A previous study demonstrated that the removal of four leaves per plant results in a moderate yield of flue-cured tobacco during the tobacco growing season9. In this study, the number of left leaves of K326 and the proportion of superior tobacco were higher, whereas the yield and economic value were lower, while the results of the Hongda cultivar were the opposite; this may indicate that the Hongda cultivar is more suited to the Honghe tobacco-planting area. However, both the yield and economic value of flue-cured tobacco were affected by the interaction between tobacco cultivar type and planting site; K326 was more suited to the JS site, and there were no significant differences between K326 and Yun 87 at the ML site, with both producing higher yields with a higher economic value.Many factors affect the quality of tobacco leaves, and as a cash crop, the yield is an important index40, thus exerting significant influence on the smoking quality of cigarettes41.The chemical components in tobacco leaves and their proportions also determine the quality of tobacco leaves42 and reduces the yield and quality of tobacco leaves43. Available nitrogen is needed to sustain full growth until flowering, which can directly influence the yield of flue-cured tobacco44, but excessive nitrogen will reduce the content of phenolic substances and lignin in plants and weaken the disease resistance of tobacco plants45. Even after the flowering stage, nitrogen overfertilization leads to a reduction in quality and economic value and gives rise to nitrogen pollution in the soil and underground water44. Overall, nitrogen contents in tobacco leaves were higher at the JS and SP sites, lower at the LX and ML sites, and negatively correlated with field rainfall and field relative humidity. Total nitrogen and alkaloid contents are used to reflect nitrogen supply capacity43. The presence of alkaloids significantly affects the taste of tobacco, and a nicotine content that is too high can increase the biting taste of tobacco46. The nicotine content was highest in the Yun87 cultivar, and nicotine contents were higher at the JS and SP sites than at the LX and ML sites. The nicotine content was positively influenced by field temperature and field sunshine hours but negatively affected by field relative humidity in this study. The nicotine content in tobacco can be increased by 1 g/kg and 3.3–3.5 g/kg by a 1.0 °C increase in the ground temperature at 0–5 cm and the air temperature, respectively47; thus, to some extent, temperature increases can stimulate an increase in nicotine content in tobacco.Nitrogen deficiency is cited as “the most common deficiency in tobacco”48. Potassium deficiency leads to a significant decline in plant growth and yield, and a low potassium content in flue-cured tobacco is always a matter of concern in tobacco production in China. Overall, the potassium content in the Yun87 cultivar did not differ significantly among the four sites, whereas potassium contents in K326 and Hongda were the highest at the SP site. The results of this study are consistent with reports that potassium content is negatively correlated with soil pH and positively correlated with AN49. However, climatic factors, not soil factors, were the significant factors influencing potassium content in this study; the potassium content was positively correlated with field sunshine hours and field temperature but negatively correlated with field rainfall and field relative humidity. Slightly acidic soil conditions, improved soil physical and chemical properties, and appropriate soil nitrogen content can promote the absorption and accumulation of potassium in tobacco, improving the potassium content. Although potassium deprivation in soil has been shown to lead to large reductions in plant growth and yields50, in our study, the yield was not influenced by AK content.The potassium content in tobacco leaves is considered an important indicator of high quality. A high potassium content in tobacco leaves can improve the combustibility, aroma, taste, and safety, and the potassium content plays a key role in the appearance and internal quality of tobacco leaves50. In our study, the K326 cultivar had the highest chlorine content, and chlorine contents were highest at the LX site, which was significantly influenced by the SCL content. Chlorine content was also positively correlated with field rainfall and field relative humidity but negatively correlated with field sunshine hours and field temperature. Chlorine in tobacco leaves is mainly absorbed from soil, while the chlorine input to the soil is primarily from water and fertilizer, mostly from previous crops and irrigation water in China51.Flue-cured tobacco is a chlorine-free crop, and a chlorine content that is too high or too low is bad for the growth of tobacco plants. When the chlorine content is too low, the tobacco leaves are easily broken, affecting the quality. Relatively large amounts of chloride have many adverse effects on the quality of tobacco leaves, resulting in effects such as a poor burning capacity, muddy appearance, and undesirable odor as well as high hygroscopicity, which causes discoloration during storage52. A sugar content that is too low or too high can influence the taste, likely resulting in a more biting or acidic taste54. Based on the analysis of our results, PTS contents in the Hongda and Yun87 cultivars did not differ among the four planting sites, whereas the PTS content in the K326 cultivar was lowest at the SP site but did not differ among the other three sites. Moreover, the PRS content in K326 did not differ among the four planting sites, whereas the PRS contents in Hongda and Yun87 were higher at ML and SP and lower at JS and LX. Sugar contents were negatively correlated with soil pH, and PRS content was positively correlated with field sunshine hours but negatively correlated with field rainfall. However, both PTS and PRS contents were positively correlated with irrigation water in Guizhou23.Sugar content is an important index used to evaluate the quality of tobacco and can also reflect the carbon supply capacity55. A high sugar/alkaloid ratio is associated with mildness, whereas a low sugar/alkaloid ratio is associated with harshness32. In our study, the PSA ratio for all tobacco cultivars was > 11, which is markedly above the upper limit of the range for this ratio, and values were lower at the JS and SP sites compared to the ML and LX sites. Furthermore, a PNA ratio closer to 1 is better for the quality of flue-cured tobacco32. The PNA ratios were all < 1 in our study, although the average value for the K326 cultivar was closer to 1. The probable cause is that the soil total nitrogen was not suited for forming optimal sugar/alkaloid and nitrogen/alkaloid ratios, similar to high total nitrogen content conditions29. These two ratios were positively correlated with field relative humidity and negatively correlated with field temperature in this study. Tobacco is meant to be flavorsome, and its quality is mainly evaluated by smoking24. The internal chemical composition of tobacco leaves will also affect the sensory evaluation of tobacco leaves and is influenced by the sugar/alkaloid equilibrium. Specifically, when the internal chemical composition is appropriate, the sensory evaluation score is relatively high.Flue-cured tobacco with a high smoke quality has an optimal sugar-to-alkaloid ratio of 6–10, with 10 being the best57. The petroleum ether extract content is influenced by the interactive effects between cultivars and ecological factors5. In our study, the petroleum ether extract content was affected by the interactive effects between soil and climatic factors and positively correlated with field sunshine hours and temperature but negatively correlated with field rainfall, field relative humidity, soil clay content, soil pH, and SOM content. On the other hand, when the starch content in tobacco leaves during harvesting is too high, it can cause an imbalance in the chemistry profile by decreasing the nicotine content58, thus reducing industrial usability. Overall, the starch content in the Hongda cultivar was the highest and was highest at the LX site. In this study, starch content decreased significantly with increasing temperature and was positively correlated with field rainfall but not influenced by soil properties.Petroleum ether extract and starch determine the quality and appearance rating of tobacco leaves50. The PTS, PRS, PTN, and PN contents can reflect the carbon supply capacity and nitrogen supply capacity52, whereas the PTK content affects quality48, and the PPE and PS contents determine the quality and appearance rating of tobacco leaves57. Among these indices, the PRS, PN, PTK, and PPE contents were positively correlated with temperature factors but negatively correlated with moisture factors, whereas the PCL and PS contents were negatively correlated with field temperature but positively correlated with field rainfall. On the other hand, although the seven soil physical and chemical properties influenced the five indices related to the internal chemical composition of tobacco leaves , soil pH was a dominant factor that influenced four indices.The ecological factors that influence the yield and chemical composition of tobacco leaves were divided into two categories: climatic factors and soil physical and chemical properties. Flue-cured tobacco is a chlorine-free crop, and the quality of tobacco leaves is closely related to the carbon and nitrogen metabolism of tobacco plants32, and tobacco quality is influenced by rainfall59. Low rainfall and relative humidity values and high sunshine hours and temperature-related indices are good for producing flue-cured tobacco of high smoking quality in southwestern China60. In this study, except for the chlorine and starch contents, the chemical composition in flue-cured tobacco was positively correlated with field temperature and field sunshine hours but negatively correlated with field rainfall and field relative humidity. Further analysis showed that at the LX site, yield and quality in the three cultivars were positively correlated with field rainfall and field relative humidity, but there are obvious differences in drought responses among the different tobacco cultivars24. To distinguish the responses of the three cultivars to drought stress, the relevant mechanisms of their stress responses should be further analyzed. The AN content also positively influenced the yield and quality of the K326 cultivar, which is line with reports that the highest tobacco yield can be obtained with high rates of nitrogen application and watering61. However, the yield and quality of the Hongda and Yun 87 cultivars were primarily affected by field temperature and field sunshine hours.RDA results showed that field rainfall, field temperature, and field sunshine hours significantly influenced the yield and quality of the K326, Hongda, and Yun87 cultivars. Similarly, different weather conditions influence the smoking quality of flue-cured tobacco52.The undulating mountains and complex and diverse landforms in the Hani-Yi Autonomous Prefecture of Honghe affect the climactic conditions in this region. Differences in latitude and altitude result in climate variations, and natural conditions differ greatly among sub-regions. The climate is very different among different tobacco-growing sites. The sampling sites belong to different eco-climatic regions to better represent the climate characteristics of the Hani-Yi Autonomous Prefecture of Honghe. Moreover, quality characteristics of tobacco leaves vary widely among different ecological regions. Among the four sampling sites, ML and LX are high-latitude and high-altitude tobacco-growing sites, with the most suitable temperature, a medium level of sunshine hours, and moderate rainfall during the field period, where plants produced higher PTS, PPE and PS contents. JS and SP are low-latitude and low-altitude tobacco-growing sites, with lower rainfall, higher temperature, and longer sunshine hours, where plants produced higher PTN and PN contents. Understanding the variations in characteristics of tobacco leaves among different ecological regions, specifically, as they pertain to which factors influence the quality characteristics of tobacco leaves at each tobacco-growing site, is important for maintaining the stability of and continuously improving local tobacco quality51. No special agronomic practices have been adopted at the flue-cured tobacco-planting sites sampled in this study. The amount of fertilizers applied often differs because the basic fertility of the soil and the demand of crops are usually considered when applying fertilizer in the field27. In addition, although the irrigation method used can affect the quality of flue-cured tobacco, the methods are not determined by the cultivars but by the sources of available water23. However, the growing time, number of left leaves, picking time, and other characteristics are also strongly correlated with cultivar type. In addition to a discussion on the effects of agronomic practices in different tobacco planting sites on the yield and quality of flue-cured tobacco cultivars in this study, we plan to carry out research in this area in the future to find the most suitable agronomic methods for particular flue-cured tobacco cultivars and provide more scientific guidance for the cultivation and production of local tobacco cultivars. Furthermore, we will also focus on the plant-soil interactions of different cultivars in the same field, which is an important mechanism in the growth of flue-cured tobacco.Finally, agronomic practices affect the yield and quality of flue-cured tobaccoIn conclusion, we found 13 variables affecting the yield and internal chemical composition of flue-cured tobacco in this study. Among them, soil properties and climatic factors influenced 7 and 11 variables, respectively. Specifically, soil pH and clay, AN, AP, and SCL contents were the main factors affecting the yield and quality of flue-cured tobacco. In addition, the economic value of flue-cured tobacco was not positively correlated with the number of left leaves or the proportion of superior tobacco. Higher temperatures and longer sunshine hours were beneficial for improving the yield of flue-cured tobacco and can increase the nitrogen and nicotine contents. However, lower temperatures, shorter sunshine hours, and higher rainfall can improve total sugar, petroleum ether, and starch contents. More importantly, the yield and quality of the K326 and Yun87 cultivars were primarily influenced by moisture factors, whereas the yield and quality of the Hongda cultivar was primarily affected by temperature factors. These results demonstrate that climatic factors were the main factors affecting the yield and quality of flue-cured tobacco.


Mitochondrial DNA (mtDNA) analysis is an important tool to assess the maternal origin and phylogeny of domestic animals. Fuzhong and Xilin buffalo are two native buffalo breeds in Guangxi, a province in the southwest of China that belongs to the major hotspots of swamp buffalo domestication centers. In this study, we sequenced the complete mitochondrial genomes of two buffalo breeds in Guangxi Province to perform the phylogenetic analysis.n = 15; Xilin buffalo, n = 25) based on the complete mitogenome sequencing. Our results revealed rich genetic diversity in the two buffalo breeds. We detected five haplogroups  in the two Guangxi buffalo breeds, and the haplogroup SB3 in the Fuzhong buffalo. Our results showed that the haplogroup SA1 was associated with the major domestication event that involved population expansion in Guangxi buffalo. In conclusion, our findings revealed a high level of maternal genetic diversity and the phylogenetic pattern of the two Guangxi buffalo breeds.Guangxi Province, in the southwest of China, is one of the putative migratory corridors or domestication centers for swamp buffalo. In this study, we investigated the evolutionary status of two Guangxi native buffalo breeds (Fuzhong buffalo,  Bubalus bubalis), a valuable species in tropical and subtropical climates, provides meat, milk, and hides [The domestic Asian water buffalo  is a very useful tool to investigate genetic diversity, phylogeny, and maternal origins of domesticated animals. However, to date, most studies have been limited to the mitochondrial D-loop region and e b gene ,6,7,8,9.e b gene ,10. Wang® UltraTM , according to the manufacturer’s recommendations. Firstly, each DNA sample was fragmented to a size of 350 bp, and then end-polished, A-tailed and ligated with the full-length adaptor to perform sequencing with further PCR amplification. Secondly, the PCR products were purified by an AMPure XP system , the size distribution of libraries was analyzed using the Agilent 2100 Bioanalyzer , and the libraries were quantified by real-time PCR. Then, the index-coded samples were clustered by the cBotCluster Generation System , according to the manufacturer’s instructions. Lastly, libraries were sequenced on an Illumina HiSeq platform.We sampled a total of 40 swamp buffalo from 2 buffalo breeds in Guangxi Province, including 15 Fuzhong buffalo and 25 Xilin buffalo. All experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People’s Republic of China. This study was approved by Institutional Animal Care and Use Committee of Northwest A&F University (permit number: NWAFAC1019). Genomic DNA was extracted from ear tissue using the standard phenol-chloroform protocol. Sequencing was performed on an Illumina HiSeq 2000 at the Novogene Bioinformatics Technology Co., Ltd., Beijing, China. For each individual, 1–15 μg of DNA was used to construct libraries using the NEBNextTo assemble the complete mitochondrial genomes (mtDNA), we mapped the sequencing reads to the swamp buffalo mitochondrial genome (NC_006295.1) using BWA-MEM v0.7.13-r1126) with the default settings 26 with t, and oneHaplotype diversity (H), nucleotide diversity (π), numbers of haplotypes, variable sites, and the average number of nucleotide differences (k) were estimated using DnaSP 5.0 . The neiIn this study, we analyzed the sequence variation of 40 complete mitogenome sequences  from two Guangxi buffalo breeds to assess their mtDNA genetic diversity, phylogeny, and maternal origin. There were 164 variable sites detected among the 40 samples, which defined a total of 28 haplotypes . The mtDNext, we estimated the mtDNA genetic diversity and haplogroup frequencies in the two Guangxi buffalo breeds . The hapSyncerus caffer (accession no. NC_020617.1) was used as an outgroup  and 2 riverine sequences; outgroup b. Phylogoutgroup a–c. HaplIn order to focus on the phylogenetics among Guangxi buffalo, a network of 40 buffalo samples was constructed c. As expcytochrome b gene [Fuzhong and Xilin buffalo are the native Guangxi buffalo breeds that are distributed in the hotspots for potential swamp buffalo domestication ,10. To de b gene ,6,7,8,9.cytochrome b gene [The phylogenetic analysis showed that all of the Guangxi buffaloes included in this study were swamp buffalo. Previous studies have identified seven frequent swamp buffalo haplogroups , together with three rare haplogroups . Wang et al. further subdivided the mtDNA subhaplogroups for SA1, SB1, SB2, SB3, and SD . In thise b gene . Furthere b gene ,8,17. The b gene . Therefoe b gene . The resIn conclusion, our findings revealed a high level of maternal genetic diversity and the phylogenetic pattern of the two Guangxi buffalo breeds. Our results showed that SA1 was the major domestication event in Guangxi buffalo.


Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in Western countries, and strongly associated with type 2 diabetes mellitus (T2DM). Several studies have shown that hypoglycemic agents are effective for NAFLD combined with T2DM. However, there is still controversy over which hypoglycemic agent is the best for NAFLD combined with T2DM patients.To systematically evaluate the efficacy and safety of hypoglycemic agents in NAFLD combined with T2DM patients.A comprehensive electronic search will be conducted by searching Web of Science, PubMed, EMBASE, Cochrane Central Register of Controlled Trials, Clinical Trials and Chinese Biomedical Medicine. All randomized controlled trials of hypoglycemic agents interventions for NAFLD combined with T2DM will be identified. Two reviewers independently screened and evaluated each included study and extracted the outcome indexes. ADDIS 1.16.8 software will be used for the network meta-analysis and STATA 14 software will be used for drawing network evidence plots and funnel plots.This network meta-analysis will provide stronger evidence for the efficacy and safety of hypoglycemic agents in the treatment of NAFLD combined with T2DM, and provide a reference for clinical application.INPLASY202070016. Since metabolic syndrome or type 2 diabetes mellitus (T2DM) is strongly associated with NAFLD, the prevalence of NAFLD has been reported to reach 70% in patients affected by T2DM.–9 Insulin resistance plays a key role in pathogenic mechanisms of NAFLD and it acts as a trigger for progression of steatosis towards steatohepatitis, cirrhosis, and end-stage complications.,11 Similarly, several articles have shown that NAFLD also substantially increased the risk of T2DM.–14 NAFLD interacts with T2DM, and insulin resistance is the main driving factor. Therefore, hypoglycemic agents have been used in the treatment of NAFLD and T2DM.,16Non-alcoholic fatty liver disease (NAFLD) is a spectrum characterized by a pathological feature of diffuse bullous steatosis of hepatocytes, may progress to non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma.–18 In China, the prices of these different drugs vary widely. Although there have been several published studies on single drug versus placebo or single drug versus another single drug, there is still no relevant systematic study on the efficacy and safety of those various hypoglycemic agents in the treatment of NAFLD combined with T2DM.–21 Therefore, there is no strong evidence to support which drug is appropriate for patients, the newer, the better; or the more expensive, the better? In this study, the efficacy and safety of multiple hypoglycemic agents will be compared to provide a bias for patients to selectively take hypoglycemic agents.At present, commonly used hypoglycemic agents can be divided into the following categories according to the different mechanisms of action: insulin and insulin analogs, biguanides, sulfonylurea secretagogues, α-glycosidase inhibitors, insulin sensitizers, dipeptidyl peptidase 4 inhibitors, sodium glucose cotransporter 2 inhibitors, and glucagon-like peptide-1 receptor agonists.22.1 The protocol has been registered on the INPLASY website (https://inplasy.com/inplasy-2020-7-0016/) and INPLASY registration number is INPLASY202070016. No ethical approval is required since this study used data already in the public domain.The systematic review protocol will be reported in accordance with the guidelines of Cochrane Handbook for Systematic Reviews of Interventions and the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocol Statement.2.22.2.1Only clinical randomized controlled trials (RCTs) published or registered but not yet published in both Chinese and English using hypoglycemic agents to treat NAFLD combined with T2DM will be selected for this study. However, studies from animal experiments, reviews, case reports, and non-RCTs will be excluded.2.2.2Participants were individuals who are clinically diagnosed with NAFLD combined with T2DM and are simultaneously treated with hypoglycemic agents. There are no restrictions on race, gender, or age. Participants with NAFLD caused by other reasons and participants with severe heart disease, liver and kidney dysfunction, mental illness, pregnant or allergies to hypoglycemic agents will be excluded.2.2.3Both groups of participants were treated with different hypoglycemic agents, or 1 group received hypoglycemic agents, while the other group received placebo, or no treatment was applied. In addition, neither group took any drugs that interfered with the outcome indicators.2.2.4The primary outcomes include the improvement in clinical efficacy and imaging markers, biomarkers of hepatic steatosis, serological indexes of hepatic fibrosis, serum NAFLD liver fat score.Secondary outcomes are mainly composed of fasting blood glucose, 2 hours postprandial blood glucose, HbA1c, serum insulin levels, aspartate transaminase, alanine transaminase, γ-glutamyl transferase, and adverse events.2.3We will search publications in the following English and Chinese database: Web of Science, PubMed, EMBASE, Cochrane Central Register of Controlled Trials, Clinical Trials and Chinese Biomedical Medicine, and the retrieval time will be set to the time of database-building to May 31, 2020. The combination of subject headings and free words will be used as a search strategy for a comprehensive electronic search. Subject headings include “hypoglycemic agents,” “non-alcoholic fatty liver disease,” “diabetes mellitus, type 2,” and “randomized controlled trials.” In Figure 2.4First, all the retrieved data will be imported into Endnote X7 software. Two researchers will independently screen and remove the literature that clearly does not meet the inclusion criteria by reading the title and abstract, and eliminate the research that does not meet the requirements, and then carefully read the full text and select the research that meet the requirements. Second, we will carefully read the full text of the remaining literature to further decide whether to include or not. Finally, the 2 researchers will independently extract the data and carefully check it by a third researcher. If there are any disagreements during the data collection process, we will reach agreement through discussion or seek advice from a third party. The data we extract include the following:(1)the basic information of the research, including the title, first author, year of publication and country;(2)the characteristics of the research, including the number of participants in the experimental group and the control group, ethnicity, and the intervention measures and treatment duration of each group;(3)outcomes indicators and data;(4)information required to assess the risk of bias.,24We will use the RCT bias risk assessment tool recommended by the Cochrane Handbook for Systematic Reviews of Interventions 5.1.0 to perform bias risk assessment and methodological quality assessment of included RCTs.2.5 = 0.05. Heterogeneity testing will be implemented to detect statistical heterogeneity between each study. If there is no heterogeneity, then network meta-analysis will be performed directly. If there is heterogeneity, then we will analyze and describe the source of the heterogeneity. The inconsistency test will be carried out using the node-split model. If P > .05, it means that there is no significant statistical difference between direct and indirect comparison, then the consistency model will be selected for analysis; otherwise, the inconsistency model will be selected.,26 We will use a sorted probability table to rank the probabilities of each intervention, further analyze the advantage and disadvantages of each intervention. We will use the STATA 14 software to draw a network diagram showing the comparisons between them and a funnel plot can be used to make a qualitative judgment of publication deviation.In this study, ADDIS 1.16.8 software will be used to perform the network meta-analysis through the Bayesian hierarchical model. For the dichotomous variables, the odds ratio will be used as an effect indicator, while for continuous variable, the weighted mean difference or standard mean difference will be used. The results of the effect analysis statistics will be presented by the estimated value and 95% confidence interval, and we will set the significance level at α2.6If necessary, we will conduct subgroup analyses on characteristics such as gender, age, race, nationality, type of hypoglycemic agents, and duration of medication to explore whether treatment effects for our primary outcomes are robust.2.7We will evaluate the publication bias by making funnel plots, of course, this is under the condition that more than 15 articles can be quantitatively analyzed. If the data in the funnel plots are symmetrically distributed, it means that there is no publication bias, otherwise we will analyze the possible reasons and give a reasonable explanation for asymmetric funnel plots.2.8We will use the Grading of Recommendations Assessment, Development, and Evaluation system to evaluate the quality of our evidence. In this system, the level of evidence quality is divided into 4 levels: high quality, moderate quality, low quality, and very low quality.3,30 There is convincing evidence that NAFLD is closely associated with T2DM, and the simultaneous prevalence of 2 diseases is a major global challenge.,31 However, approved treatments for NAFLD combined with T2DM are limited.Studies have shown that NAFLD is a multi-system disease that can affect multiple extrahepatic organ systems and interact with multiple metabolic/endocrine and pro-inflammatory pathways.Hypoglycemic agents have been widely used in the first-line treatment of NAFLD combined with T2DM, and their efficacy and safety should be guaranteed. In addition, hypoglycemic agents may be drugs that patients are required to take for life, so it is particularly necessary to provide patients with more information on the efficacy and safety of hypoglycemic agents in the treatment of NAFLD combined with T2DM, so as to better help patients control disease progression and reduce treatment costs.In conclusion, this study will conduct a systematic review and network meta-analysis of relevant RCTs, and will comprehensively assess whether hypoglycemic agents are beneficial to NAFLD combined with T2DM. In addition, this study will compare the advantages and disadvantages of various hypoglycemic agents, and provide higher quality evidence for the selective use of hypoglycemic agents in NAFLD with T2DM patients and guide clinical practice.Conceptualization: Su-Tong Liu, Kai-Qi Su.Data curation: Li-Hui Zhang, Wen-Xia Zhao.Methodology: Su-Tong Liu, Kai-Qi SuSoftware: Kai-Qi Su, Ming-Hao Liu.Supervision: Su-Tong Liu, Wen-Xia Zhao.Writing – original draft: Su-Tong Liu, Kai-Qi Su, Li-Hui Zhang, Ming-Hao Liu, Wen-Xia Zhao.Writing – review & editing: Su-Tong Liu, Kai-Qi Su.


This study aimed to determine the accuracy of the Breath-Alert™ portable breath meter (BA) for the detection of halitosis in children and adolescents, considering the organoleptic test (OT) as the gold standard in this assessment.A cross-sectional study was conducted on 150 children (aged 6-12 years). OT was performed by three independent examiners on a single occasion, obtaining three scores of 0-5 points on the Rosenberg’s organoleptic scale. The median of the three evaluations for each child was used for analysis. BA was used according to the manufacturer’s instructions, with breath odor scored from 0-5 points. Scores ≥2 on both tests were considered indicative of halitosis.A total of 26 (17.3%) and 23 (15.3%) children were detected with halitosis on the OT and BA tests, respectively. The sensitivity and specificity of the BA scores for the detection of halitosis were 80.76% and 98.38%, respectively. The positive and negative predictive values for BA were 91.3% and 96.06%, respectively.In the present study involving children, who require fast, practical examinations, BA proved to be an auxiliary tool to OT for the detection of halitosis in the practice of pediatric dentistry, demonstrating high sensitivity and specificity. Halitosis is an imbalance in which breath odor is altered in an unpleasant manner, causing a significant social and psychological disadvantage for affected individuals . GloballThe foul odor in exhaled air is caused by volatile sulfur compounds (VSCs) produced by anaerobic Gram-negative bacteria  on substThe literature describes two main methods for the assessment of halitosis: subjective (organoleptic) and objective (gas chromatography or a sulfide monitor) ,13. The The Breath-Alert™ portable device (BA) has been increasingly employed in clinical practice due to its ease of use and low cost -19. ThisAs a proper diagnosis is of the utmost importance to determine effective solutions for the treatment of halitosis , it is eThis study received approval from the Human Research Ethics Committee of the Federal University of São Paulo (certificate number: 610.481). All parents/guardians and children received clarifications regarding the objectives and procedures of the study and signed statements of informed consent.A prospective cross-sectional study was conducted with children scheduled for dental treatment at the pediatric clinic of the pediatric dentistry specialization course of the Paulista Association of Dentists . Children aged 6-12 years in the mixed dentition phase  were included in the study. The exclusion criteria were tonsillitis, sinusitis, neurological, psychiatric, or behavioral disorders, and the use of medication. A total of 167 children were examined, and 150 of them who met the eligibility criteria were included in the study.The evaluations were performed in two sessions. In the first session, the children and caregivers answered a questionnaire addressing personal data, general health, oral health, and lifestyle and eating habits. The children and caregivers were then instructed to avoid eating spicy and/or aromatized food 24 hours prior to the session, and eat an adequate meal 3 hours prior to the session, followed by habitual tooth brushing and flossing, without the use of mouthwash, breath mints, or chewing gum, prior to the second session. Children were also instructed not to use deodorants, perfumes, cosmetics, and creams that could "confuse" the judges.In the second session, the children were clinically evaluated for halitosis using OT and BA.To minimize subjectivity, OT was performed by three different examiners in the same session, with each examiner blinded to the other assessments. Thus, each child was evaluated three times independently, obtaining three scores ranging from 0-5 points on the Rosenberg’s organoleptic scale  20-22).-22.20-22BA was used according to the manufacturer’s instructions and disinfected after each use. The device was shaken four or five times prior to each use to eliminate any residual odors. A “beep” was emitted on opening the upper compartment of the device, and a second “beep” was emitted when the volunteer blew into the frontal air entrance (air flow passage). After a third “beep,” the breath odor was measured and scored on a scale of 0-5 points. When the letter “E” appeared, indicating an error, the procedure was repeated . A scoreThe sample size was calculated considering the 52% prevalence rate reported by the Brazilian Halitosis Association, a 5% significance level, and 8% margin of error, leading to a minimum sample of 150 children. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the BA test in comparison to OT. Data analysis was performed using IBM SPSS (Subscription version 05-2017) for the MAC (Apple Inc.) operating system.The sample was composed of 150 children (54% girls and 46% boys) with a mean age of 9.1±1.4 years (range: 6.1-12.2 years). Twenty-six children (17.3%) were classified as having halitosis when assessed by OT. However, when assessed using BA, 23 (15.3%) were classified as having halitosis and 2 (1.3%) were false positives.OT is widely used since it is inexpensive, easy to administer and does not require any technological device ,17,22. TAlternatives to OT include the OralChroma™ gas chromatograph, Halimeter™, and Breath-Alert™. Gas chromatography is the most effective objective method to compare the efficacy of halitosis tests ,25, as iIn the present study, BA demonstrated high sensitivity (80.76%), although it failed to diagnose some children with halitosis that were positive on OT. This divergence may be because VSCs are the main contributors to bad breath, but other organic compounds are also found in exhaled air . PortablTo improve the reliability of diagnosis, halitosis should be assessed using two different methods , with OTIn the present study, BA was found to be useful for the rapid detection of halitosis in daily pediatric dental practice, demonstrating high sensitivity and specificity.Guedes CC was responsible for the conceptualization, data curation, investigation, methodology, visualization, and manuscript writing. Bussadori SK was responsible for the investigation, methodology, and visualization. Garcia ACM was responsible for the data curation, investigation, and visualization. Motta LJ was responsible for the formal analysis, software resources, and visualization. Gomes AO was responsible for the software resources and visualization. Weber R was responsible for the formal analysis and software resources. Amancio OMS was responsible for the conceptualization, data curation, methodology, and manuscript writing.


Background: The unique immunomodulatory capacity of helminth parasites has been investigated as a novel strategy in the prevention of allograft rejection after transplantation. This review was conducted to fully evaluate the specific effects of helminth therapy on allograft survival reported in published studies of animal models of allogeneic transplantation.Method: Following PRISMA protocol guidelines, a literature search was conducted using PubMed, MEDLINE via OvidSP, along with additional manual searches of selected reference lists. Publications describing helminth intervention within allograft transplantation models were screened for relevance to eligibility criteria. Primary and secondary outcomes were extracted using standardized data collection tables. The SYRCLE risk of bias assessment tool was used for quality assessment. Due to heterogeneity of study designs, meta-analysis could not be performed; rather outcomes are presented as a narrative synthesis with concept mapping. This review was registered in PROSPERO with ID: CRD42018097175.Results: The literature search generated 1,443 publications, which after screening for relevance to the eligibility criteria yielded 15 publications for qualitative analysis. All 15 publications reported improvement to allograft survival as a result of helminth therapy. This prolonged allograft survival was not significantly different when helminth-derived products were used compared to live infection. However, the extent of positive impact on allograft survival was noted to be dependent on study design factors, such as the chronicity of the live helminth infection, allograft type and the species/genus of helminth selected.Conclusion: Both live and product-based helminth therapy have potential applications as novel immune regulators or adjuncts for the prevention of allograft rejection. However, there were differences in efficacy between different worms and preparations of worm-derived products. Therefore, further studies are required to determine the most appropriate worm for a specific allograft, to elucidate the optimal dose and route of administration, and to better understand the modulation of immune responses that can mediate tolerance. Trichinella spiralis occurs through ingestion of meat that is contaminated with the larval stage of the parasite. In the case of schistosomiasis, infection occurs after the parasite cercaria is released from its intermediate snail host and burrows through the skin of its mammalian host. Larvae migrate through the lung and heart before they mature in the liver. Although infection with Nippostrongylus brasiliensis also occurs through subcutaneous infection, unlike the schistosomes that ultimately inhabit the mesenteric blood vessels, N. brasiliensis larvae exit the circulation into the lungs and are coughed up and swallowed, eventually residing in the intestine of its chosen mammalian hosts. Despite such diversity in the morphology and life cycles of individual helminths, this group of parasites induces a stereotypical Th2 immune response, characterized by the presence of cytokines IL-4, IL-5, IL-13, and IL-10, antibody isotypes IgG1, IgG4, and IgE, and expanded populations of eosinophils, mast cells, basophils and M2 macrophages (Parasitic worms (helminths) are a highly divergent group of macro-pathogens consisting of two major phyla: the Nematoda (or roundworms) and the Platyhelminthes , with the latter consisting of both trematodes (flukes) and cestodes (tapeworms) . ReflectIt has been proposed that this specific profile of immune response has evolved as a mechanism to regulate the extensive tissue damage caused by these macro-pathogens as they migrate through the host, resulting in a dynamic co-evolution between the host and the parasite , 3. IndeIn contrast to micro-pathogens, such as bacteria, protozoa, and viruses, most helminths do not replicate in their mammalian hosts. Instead, the infective stages must establish infection and then grow to sexual maturity, producing eggs or live offspring for transmission to the next host. Therefore, maintaining host fitness is in the best interest of the parasite. This is supported by the observation that in addition to host signals of damage, a number of parasite-secreted compounds also modulate host immune responses to ensure a positive outcome for both host and parasite , 5.Frequently, helminth parasites establish chronic infections and if untreated can persist for decades in the tissue of their hosts. During these long-term infections, it has been noted that the dynamics of the T cell response changes over time. To prevent excessive tissue repair, which may result in fibrosis-related pathology, the Th2 response often declines during chronic infection and is replaced with the development of regulatory pathways, including the differentiation of regulatory T cells (Tregs). This transition, actively initiated by both host and parasite signals, ensures the fine-tuning of the parasite-driven immune response to restrain pathological outcomes , 6.This potent modulation of host immunity by helminths has additional unintended consequences, resulting in the regulation of a variety of inflammatory conditions caused by a dysregulated immune response, such as rheumatoid arthritis, multiple sclerosis, ulcerative colitis, type 1 and 2 diabetes, and sepsis –10. In bIn light of this immunomodulatory capability, many have suggested extending the use of helminth therapy to the prevention of allograft rejection. Successful allograft transplantation is continually plagued by the challenge of both acute and chronic cell mediated allograft rejection . This prTo date, there has been no single comprehensive study on the use of helminth therapy as an adjunct to traditional immunosuppressive therapy. In particular, the analysis of the impacts of study design, the choice of parasite, and importantly, the difference between live and product-based helminth therapy on the survival of allografts has not been determined. Therefore, this systematic review aims to apply these considerations to the qualitative analysis of published studies to better characterize the effects of live and product-based helminth therapy on the survival of allogeneic transplants.This systematic review was conducted according to the provided Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines (PRISMA)  and regi* OR nematode OR trematode OR cestode OR worm* OR “parasitic worm*” OR schistosome* OR trichuris OR nippostrongylus OR fluke OR tapeworm OR hookworm) AND . Publications were screened for duplicates and for relevance to outlined criteria. A secondary reviewer provided resolution to any discrepancies in publication relevance.Publications were gathered from database searches of PubMed and Medline via OvidSP. Additional publications were compiled from reference lists of literature reviews and included studies until January 2020. The following search terms were utilized for both databases: .Bias was assessed by two reviewers using an adapted form of the SYRCLE risk of bias assessment tool, a version of the Cochrane RoB tool . Items 1Due to heterogeneity of the publications, meta-analysis was not applicable for this systematic review. Studies are instead presented in narrative synthesis, detailing the effect of helminth therapy on allograft survival. The primary outcomes of graft survival (days post-transplantation) was reported in addition to supplementary data presented in each study, including immunohistochemistry, flow cytometry and serum cytokine detection. Differences in graft survival for each allograft presented, were compared by considering the helminth genus and species, allograft type, and parasite burden utilized. In addition, the effect of live and product-based intervention type was considered for its impact on graft survival. These were then presented as concept maps to visualize the impact of such study design characteristics on the primary outcome.As shown in The characteristics of the included studies are summarized in n = 9) (n = 3) (n = 1) (n = 1) (Echinococcus multilocularis (n = 2) (Nippostrongylus brasiliensis (n = 2) (Schistosoma mansoni (n = 2) (Paragonimus westermani (n = 1)   (n = 9) (n = 2) (n = 5) (n = 1) (n = 1) (n = 4) (n = 5) (n = 3)  –31, hear (n = 3) –34, kidn (n = 1) , and liv (n = 1) . One stu (n = 1) . Helmint (n = 2) , 36, Nip (n = 2) , Schisto (n = 2) , 33, Par (n = 1) , Trichin (n = 6) , 30, 37, (n = 2) , 31. Hel (n = 2) , 33 trea (n = 2) , 31, 35. (n = 5) , 29, 35, (n = 1) , or puri (n = 4) , 36, ora (n = 5) –28, 37,  (n = 3)  or intran = 2) (n = 7) (n = 2) (n = 1) (n = 1) (n = 3)  for control vs. treatment for the vast majority of studies. One publication did not report the time until graft rejection . Graft rn = 2) , 24, nec (n = 7) , 31, 36, (n = 2) , 37, los (n = 1)  increase (n = 1) , or cess (n = 3) , 33, 37.n = 4) (n = 6) (n = 1) (n = 3) (n = 1)  , 35–37,  (n = 6) –37, dire (n = 1) , serum c (n = 3) , 36, 37, (n = 1) , 33, 37.The results of the SYRCLE bias assessment of the 15 publications included in this systematic review are reported in Most of the publications were unclear on their reporting of randomization and blinding. Reports of random housing were unclear in 53% of publications and blinding of outcome assessors was unclear in 40%. These two features were deemed in this review to be of minimal importance for the assessment of allograft rejection. Importantly, 93% of included publications reported similar baseline characteristics, a necessary component for comparing animals in transplantation studies. Additionally, 87% of publications were free from other sources of bias.All 15 publications reported significant improvements to allograft survival with the administration of both live and product-based helminth therapy . AlteratT. spiralis larvae within a skin graft model  within the graft tissue in response to both helminth infection and administration of helminth-derived products  and a parallel increase in Th2/Treg cytokines (IL-4 and IL-10) –37. Suppproducts , 33, 37.products , 33.T. spiralis and T. pseudospiralis larvae produced similar improvements to allograft survival in comparison to control  than larvae given 7 days before (17.2 days)   . As helmcontrols . Increascontrols .N. brasiliensis showed the most significant prolongation of graft survival   .T. spiralis larvae by the natural route of infection  induced no difference in graft survival when compared to that induced by a single IP injection of 0.2 mg of a soluble protein extract of the same parasite  or a soluble worm extract (prepared from 100 mg larvae) ; T. pseudospiralis . In contrast, another skin graft study which tested the same parasite species showed that live infection of either species  resulted in extended allograft survival to 24 days whilst the IP administration of soluble worm extract (50 μg) did not significantly extend graft survival past that of the control group (day 8)  of either native secretions or a soluble extract of the worm were less effective or ineffective, respectively, in preventing the rejection of skin grafts compared to studies where either higher doses (designed to imitate the presence of live worms) or live infection was used. This observation was supported by a study in a kidney allograft model .A direct comparison between live infection and the administration of helminth-derived products was specifically investigated within four separate studies , 31, 35.parasite . Both prparasite . SimilarT. spiralis larvae on both heart and skin allografts (p <0.01). Within the skin model, allograft survival was limited to 13.67 days in comparison to control (p <0.01). The graft survival duration for other solid organ (heart and liver) transplants were similarly impacted by helminth infection. Two studies investigating the effect of E. multilocularis live therapy on both liver and heart allografts (p <0.05) and heart  allografts survived to a similar extent in each study. These comparisons stand as evidence for the differences in allograft survival being influenced by the type of tissue being transplanted.Differences in the type of allograft model used also impacted on the extent of allograft survival. The smallest improvement to allograft survival was seen with skin allografts. This effect was well-modeled within one investigation of the effects of 300 lografts . Whilst lografts , 36 wereThis systematic review has determined that in all 15 publications allograft survival was significantly improved in helminth-treated groups, with all studies reporting marked decrease in graft necrosis. The positive impacts of helminth therapy on allograft survival were clearly seen across multiple animal models of allogeneic transplantation. The finding that both live infection and the administration of helminth-derived products were both effective at preventing allograft rejection, is consistent with a previous literature review of this topic  and suggT. spiralis, with the heart surviving 10 days longer than the skin graft , which likely impacted on the ability of the parasite to fully modulate the immune response as expected.The importance of inducing the correct immune response to facilitate transplant survival is also illustrated when comparing the outcomes of infection with different genera of parasites. On the surface it appears that infection with live  mansoni , 27. HowT. spiralis or T. pseudospiralis to the IP administration of soluble extracts of worms (50 μg) and to an IP injection of the native secretions of the parasite (50 μg) (p <0.01) improvement compared to the same dose of soluble extracts prepared by either homogenizing the worms (8 days) or the control group (7 days). To properly compare the native secretions of the parasite to the live infection, a dose which more accurately represents the quantity of proteins secreted by an equivalent number of larvae should be administered to the transplant recipient.Understanding the biology of the parasite to better exploit its immune modulating power becomes even more critical in the selection and analysis of the efficacy of helminth-derived products. This is most evidently demonstrated in a skin graft study that compared the impact of live infection with either  (50 μg) . It is a (50 μg) . In cont (50 μg) . AlthougS. mansoni pentasaccharide LNFP-III were required to extend median graft survival of heart transplant compared to controls (S. mansoni, the production of which coincides with the development of a potent Th2/Treg immune response in this parasite infection (P. westermani, as only a single IP injection was required to provide the greatest protection to the graft across all of the studies examined, extending the survival of a skin graft to 86 days beyond the control animals (An alternative explanation for the reduced efficacy of the native secretions compared to live infection is that the continued presence of the parasite may be necessary to elicit the regulatory immune response in the host , 49. Howcontrols . This monfection . The posnfection . Perhaps animals . Further animals  suggestiThe review was restricted by the lack of immunological evidence presented in each paper. Of the 15 publications evaluated, nine provided the primary outcome of allograft survival with no additional supporting evidence of immune profiling. This placed limitations on the conclusions that could be drawn, meaning that similarities and differences between therapies on allograft survival could only be assumed based on our current knowledge of transplant and parasite immunology. Additionally, due to the heterogeneous nature of all publications meta-analysis could not be performed, and thus a subjective qualitative synthesis was used for analysis. To reduce the subjective nature of this analysis, input from multiple reviewers was gathered when performing the narrative synthesis. Despite this, lack of statistical evidence does preclude the application of this review to influence the development of helminth-based therapies for enhanced allograft survival. In future, further publications with similar study designs may allow for meta-analysis to validate the conclusions found in this review.Despite the limitations, this review provides comparative evaluation of the effects of helminth therapy on allograft survival across multiple models of allogeneic transplantation. While variations in efficacy were most notably linked to the timing and dose of helminth therapy, and to the choice of allograft model, it was clear that all publications reported improvements to allograft survival with the administration of both live and product-based helminth therapy. It is evident that a better understanding of the immune changes induced by the parasites and their products will be essential to developing the most effective therapeutic strategy for specific allografts. Furthermore, to elucidate which parasite-derived molecules are likely to be most effective, a more appropriate analysis of these compounds is required, with consideration for their known or predicted biological function in the host. Nonetheless, the evidence presented here supports the proposition that helminth therapy represents a viable treatment form of immunosuppressive therapy. By careful selection of the helminths or helminth products that are best suited to particular allograft types and consideration of the required dose/chronicity to induce the most appropriate immune response, an optimal protocol can be achieved to provide the most significant immunomodulation to promote successful allograft survival .All datasets presented in this study are included in the article/WH conceived the idea. MK searched the literature and drafted the manuscript. WH, HB, and SD commented on the structure of manuscript, provided the critical intellectual input, and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Regarding the repair of vertebral compression fractures, there is a lack of adequate biomechanical verification as to whether only half of the vertebral body and the upper and lower intervertebral discs affect spinal biomechanics; there also remains debate as to the appropriate length of fixation.A model of old vertebral compression fractures with kyphosis was established based on CT data. Vertebral column resection (VCR) and posterior unilateral vertebral resection and reconstruction (PUVCR) were performed at T12; long- and short-segment fixation methods were applied, and we analyzed biomechanical changes after surgery.Range of motion (ROM) decreased in all fixed models, with lumbar VCR decreasing the most and short posterior unilateral vertebral resection and reconstruction (SPUVCR) decreasing the least; in the long posterior unilateral vertebral resection and reconstruction (LPUVCR) model, the internal fixation system produced the maximum VMS stress of 213.25 mPa in a lateral bending motion and minimum stress of 40.22 mPa in a lateral bending motion in the SVCR.There was little difference in thoracolumbar ROM between PUVCR and VCR models, while thoracolumbar ROM was smaller in long-segment fixation than in short-segment fixation. In all models, the VMS was most significant at the screw-rod junction and greatest at the ribcage–vertebral body interface, partly explaining the high probability of internal fixation failure and prosthesis migration in these two positions. Thoracolumbar vertebral compression fractures are common in orthopedics; these fractures include osteoporotic fractures in the elderly and fractures resulting from falls or car accidents. If not treated in a timely fashion, they may develop into old fractures, accompanied by kyphotic deformities. The symptoms of old vertebral fractures with kyphotic deformities include persistent low back pain, sometimes accompanied by spinal cord compression symptoms. Conservative treatment of old vertebral compression fractures with kyphotic deformities is often not effective; therefore, treatment is often surgical . For sevComputed tomography (CT) images of normal subjects were imported into computer modeling software to establish a model, thereby simulating various surgical osteotomy modes, restoring vector balance, and installing a screw-rod system. Finally, we used finite element analysis software to calculate and compare biomechanical parameters.We performed a finite element analysis of patients treated between 1 January 2020 and 1 June 2020 at the Department of Spinal Surgery, Tianjin Hospital, Tianjin University.Volunteers were recruited from the Department of Spinal Surgery, Tianjin Hospital, Tianjin University. A 40-year-old man, height 176 cm, and weight 72 kg underwent spinal CT scan with three-dimensional reconstruction and MRI examination to rule out other spinal diseases. After obtaining consent from the patients and their families, informed consent forms were signed following the relevant regulations and submitted to the Ethics Committee for approval.We obtained 128-row, 256-slice GE spiral CT thin-section scans  with a slice thickness of 1 mm and resolution of 512 × 512 to obtain sagittal two-dimensional tomographic images of the T8–L3 vertebral body, and files were saved in DICOM format.DICOM images were imported into Mimics 20.0  to create a 3-dimensional (3D) vertebral surface model from T10 to L2 that were stored as STL format files; 3-Matic 12.0 software  was used after wrapping, smoothing, and removal of excess triangulars. Data were imported into Geomagic Studio 12.0  for solidification.Hypermesh  was used to grid and construct the intervertebral disc, bone, and ligament structures, in which the intervertebral disc was composed of annulus fibrosus matrix, nucleus pulposus, annulus fibrosus fibers, and upper and lower endplates; the nucleus pulposus accounted for 43% of the total intervertebral disc . The surWe fabricated four fixation models using various screw combinations. The pedicle screws inserted into T11 and 12 measured 6.0 × 40 mm, and the pedicle screws inserted into L1 and two vertebral bodies measured 6.5 × 45 mm. The LVCR model refers to the removal of the whole T12 vertebral body and T11/12, T12/L1 intervertebral disc, the application of a cylinder of 18-mm diameter and 1-mm thickness to simulate a titanium cage fixed in the vacant position of the vertebral body, with cancellous bone placed in the middle. Pedicle screw fixation was applied at T10, T11, L1, and L2. SVCR refers to the removal of pedicle screws at T10 and L2 based on LVCR. LPUVCR refers to removing T12 right vertebral plate, facet joint, and vertebral body, T11/12, T12/L1 right intervertebral disc. A cylinder with a diameter of 10 mm and a thickness of 1 mm was used to simulate the titanium cage. The cage was fixed at the vacant position of the vertebral body, and the cancellous bone is placed in the middle for testing. Pedicle screw fixation was applied at T10, T11, L1, and L2. SPUVCR refers to the removal of pedicle screws at T10 and L2 on top of LPUVCR  ROM of the overall fixation (T10–L2); (2) von Mises stress of the instrumentation system; and (3) stress magnitude and distribution of the inferior endplate of T11 and superior endplate of L1. These measures were chosen to assess the role of various vertebral body reconstruction methods in the overall fixation system. Because only one subject was modeled, statistical analysis was not performed in this study.The model was determined to be reasonable by comparison with biomechanical experiments and finite element models. Using the same loading conditions, the ROM values of the complete model at the moment of 7.5 N were consistent with those of previous studies –8. This Compared with the intact model, ROMs decreased in all fixed models under loading, with LVCR decreasing the most, forward flexion decreasing by 88.8%, extension decreasing by 97.3%, left lateral bending decreasing by 89.6%, right lateral bending decreasing by 90.8%, left lateral rotation decreasing by 88.8%, and right lateral rotation decreasing by 88.9%. SPUVCR decreased the least, with 51.2% decrease in forward flexion, 33.8% decrease in extension, 17.6% decrease in left lateral curve, 14.6% decrease in right lateral curve, 78.8% decrease in left rotation, and 79.1% decrease in right rotation  were significantly smaller than those of short-segment fixed models (SVCR and SPUVCR), while the ROM of the VCR model was relatively smaller than that of the PUVCR model; however, the difference was not significant. This finding suggests that the thoracolumbar segment's ROM is greatly affected by fixation; however, it has little relative relationship with the amount of vertebral body and soft tissue removed.We found that the site with the largest VMS in internal fixation was at the screw-rod connection site in all models, explaining why internal fixation is prone to failure at the screw-rod connection site. Similar findings were reported previously –23. PUVCIn contrast, in long-segment fixation, the PUVCR titanium cage’s stress increased by 4.5%, 1.2%, and 25.2% in forward flexion, extension, and rotation, respectively, compared with VCR. In the position of the superior and inferior endplates, the stress in the short segment was significantly greater than the stress in the long segment, and this may explain why the short segment is more prone to subsidence of the anterior prosthesis. Nevertheless, the amount of vertebral body removal did not appear to affect the change of the area of stress concentration, whether it was the VCR model  or the PUVCR model . The site with higher stress was located in the contact position between the titanium cage and the endplate, suggesting that the prosthesis settlement could occur at the contact site.When performing surgery, clinicians should pay more attention to the screw-rod junction and the contact position between the titanium cage and the endplate to prevent the fracture of the screw-rod and the settlement of the titanium cage. Simultaneously, a long-segment fixation can be applied to enhance the stability of the spine. In terms of surgical methods, both VCR and PUVCR have similar fixation strengths and can achieve adequate fixation. However, PUVCR is relatively more straightforward to operate than VCR; the operation time is shorter, and there is less blood loss.This study has some limitations. First, we used CT scans of normal subjects to construct the old thoracolumbar fracture model, which may have impacted the test. Second, we only used one person to establish the model. Due to individual differences, there may be structural variations in vertebral bodies and ligaments. Third, our test was not verified using cadaver testing. In future studies, model reconstruction using several patients can be further performed, resulting in more objective results. The biomechanics of cadaveric specimens can also be used as a supplement to the finite element analysis.Compared with the VCR model, the thoracolumbar ROM of PUVCR was not significantly different, while the thoracolumbar ROM was less than that of short-segment fixation after long fixation. Except for short-segment rotations, the stresses were more remarkable for the PUVCR instrumentation systems than for the VCR. In the stress distribution, the screw-rod junction had the largest VMS. In contrast, the cage and vertebral body interface had the largest VMS, and this partly explained the high probability of internal fixation failure and prosthesis migration in these two positions.


KAT6B gene. It is characterized by genital abnormalities, patellar hypoplasia/agenesis, flexion contractures of the hips and knees, corpus callosum agenesis with microcephaly, and hydronephrosis and/or multiple renal cysts. More than half of patients with GPS have congenital heart defects, mostly atrial and/or ventricular septal defects, patent foramen ovale, and patent ductus arteriosus. We report a case of a Japanese neonate with a de novo heterozygous c.3769_3772delTCTA pathogenic variant in the KAT6B gene who presented with a cardiac intramural cavity of the ventricular septum at birth. The cavity unexpectedly disappeared at 1 month of age, but trabecular septal thinning and flash remained. The features of the cavity were not consistent with those of congenital ventricular diverticulum or aneurysm, and its identity and prognosis are still unclear. Because patients with GPS may exhibit various forms of cardiac malformation, careful cardiac examination and follow-up are required from birth in cases of suspected GPS.Genitopatellar syndrome (GPS) is a rare autosomal dominant disorder caused by de novo pathogenic variants in the  KAT6B gene in 2012 [KAT6B is located at the 10q22.2 region and encodes lysine acetyltransferase 6B, a component of the histone H3 acetyltransferase complex. The KAT6B pathogenic variants in the distal and proximal parts of exons 17 and 18 (the last exon), respectively, are known to cause GPS [Genitopatellar syndrome  is a rare syndrome characterized by genital abnormalities, patellar hypoplasia/agenesis, flexion contractures of the hips and knees, corpus callosum agenesis with microcephaly, and hydronephrosis and/or multiple renal cysts , and the in 2012 , 5. KAT6Congenital heart defects are present in more than 50% of patients with GPS , 6. MostThe patient is the first son of nonconsanguineous Japanese parents with no family history of congenital anomalies. At the time of his birth, his father and mother were 34 and 25 years old, respectively. Intrauterine growth retardation and hydronephrosis were observed at 22 weeks of gestation, and ventricular enlargement at 36 weeks of gestation was noted via fetal ultrasonography. Amniocentesis revealed a fetal karyotype of 46,XY. The patient was born at 38 weeks and 2 days of gestation by normal vaginal delivery. His birth weight, height, and head circumference were 2,203 g (−1.98 SD), 47.0 cm (−0.67 SD), and 29.8 cm (−2.37 SD), respectively. Apgar scores were 6 at 1 min and 8 at 5 min. He was treated in the neonatal intensive care unit for respiratory distress.He had dysmorphic features including microcephaly, bulbous nose, wide nasal base, bitemporal narrowing, micrognathia, and retrognathia Figures . AdditioKAT6B were amplified by polymerase chain reaction (PCR), and Sanger sequencing was performed using a PCR Thermal Cycler Dice Gradient Tp600  and an Applied Biosystems 3130 DNA analyzer . Mutated and wild-type alleles were separated by subcloning the targeted PCR product into pUC19 DNA . In exon 18, a de novo 4 bp deletion was detected, c.3769_3772delTCTA, leading to a frameshift and premature stop codon (p.Lys1258Glyfs∗13) , and written informed consent was obtained from his parents. Subsequently, exons 17 and 18 of  Figures .We encountered a case of GPS with a unique cardiac muscle structure. This malformation showed the following four features: the cavity was noted inside the ventricular septum, the cavity had narrow communication to the right ventricle, the cavity wall showed dyskinesis, and the cavity resolved at 1 month of age. This cavity looked similar to congenital ventricular diverticulum (CVD) or congenital ventricular aneurysm (CVA), but its features were not fully consistent with those of CVD or CVA. Therefore, we named this malformation “cardiac intramural cavity” in the present report. The cardiac intramural cavity, as well as CVD and CVA, has not been described in other patients with GPS.CVD and CVA, recently often referred to as congenital ventricular outpouchings (CVOs) , 9, are The location and change in the size of the cardiac intramural cavity are also quite different from those of typical CVOs. Most CVOs are located at the apex or free wall of the ventricle, most frequently on the left ventricular apex , 13. OhlKAT6B, which was demonstrated to produce a truncated protein leading to a gain-of-function effect, has been previously reported in five other patients with GPS [The identified c.3769_3772delTCTA pathogenic variant in with GPS , 5, 15. with GPS , 5, and After the cardiac intramural cavity disappeared, the thinning and flash of the wall of the trabecular ventricular septum, complete right bundle branch block, and right axis deviation remained in our patient. He has been asymptomatic until now, but his long-term prognosis is still unclear. Similar to CVD and CVA, it may cause cardiac adverse events such as arrhythmia, embolism, ventricular wall rupture, congestive heart failure, and sudden cardiac death . TherefoIn summary, we encountered a case of GPS presenting with a cardiac intramural cavity of the ventricular septum at birth. The features of the cavity were not fully consistent with those of CVD and CVA in terms of the communication width to the ventricle, contractility, location, and change in size. Moreover, the identity and prognosis of the cavity remain unclear. Because patients with GPS may exhibit various forms of cardiac malformation, careful cardiac examination and follow-up are required from birth in cases of suspected GPS.