El Niño has been recorded to change its properties since the 1980s, characterized by more common extreme El Niño and Central Pacific (CP) El Niño events. However, it is still unclear whether such change is externally forced or part of the natural variability. Here, we find that the frequency of the extreme and CP El Niño events also increased during the period 1875–1905, when the anthropogenic CO2 concentration was relatively lower, but with a positive phase of the Atlantic Multidecadal Oscillation (AMO). Models and palaeoclimate proxies reveal that a positive AMO enhances the zonal sea surface temperature gradient in the CP, which strengthens zonal advective feedback, favoring extreme and CP El Niño development. Moreover, we estimate that internal variability contributed to ~65% of the increasingly extreme and CP El Niño events, while anthropogenic forcing has made our globe experience ~1 more extreme and ~2 more CP events over the past four decades. This study finds that the abnormally high frequency of extreme El Niño and central Pacific El Niño events over the past four decades can be attributed to the synchronized effects of greenhouse warming and internal variability. Extreme El Niño events have occurred frequently in the past 40 years, at a rate of one event per 13 years, usually causing reorganizations of atmospheric convection and inducing severe climatic disruptions around the globe, such as disastrous floods and droughts across the Pacific region7. At the same time, the Central Pacific (CP) El Niño10 , which is characterized by the peak ocean warming in the central equatorial Pacific, has become more common16 and the canonical Eastern Pacific (EP) El Niño, which is characterized by the strongest sea surface temperature anomalies (SSTA) located in the far eastern equatorial Pacific, has become less frequent12. The movement of the sea surface temperature (SST) anomaly center shifts atmospheric circulation and convection, causing different climate impacts around the globe17.El Niño, the warm phase of the most dominant interannual climate variability, has changed its behavior since the 1980s7 and CP El Niño variability increases14 under greenhouse warming and have suspected that the observed El Niño behavior changes could be a consequence of anthropogenic warming. With the buildup of greenhouse gases, the eastern equatorial Pacific warmed faster than the surrounding regions, and the thermocline in the equatorial Pacific flattened, facilitating more occurrences of extreme El Niño and CP El Niño events14. The enhanced response of atmospheric vapor to air temperature under greenhouse warming also increases the extreme El Niño18. Multiple palaeo-El Niño proxies also provide evidence for the impact of the climate change on the observed change in El Niño properties. These include enhanced El Niño variability during the late twentieth century relative to the preindustrial period21 and more frequent CP events in recent decades relative to the preindustrial era22. In addition to anthropogenic forcing, natural variability also plays a role in the change in El Niño properties24. Preindustrial model simulations show that El Niño characteristics exhibit strong interdecadal and intercentennial modulation in the absence of external forcing23. Palaeo-El Niño reconstructions also show a wide range of El Niño variance over the past 7000 year21 and find the El Niño variabilities in both the early 1900s and recent decades are relatively higher than preindustrial levels25, highlighting the role of internal variability. Observations and preindustrial-control model simulations show that Atlantic Multidecadal Oscillation (AMO), a long-lived basin-wide warming or cooling in the Northern Atlantic that generally persists for 60–80 years, could modulate El Niño amplitude28 and El Niño type30.Several studies using climate models have projected that El Niño amplitude increases24 or a consequence of global warming14. In order to investigate this, we can analyze past El Niño records to see whether there are other periods with frequent extreme El Niño and CP El Niño occurrences like the past 40 years, but without strong anthropogenic forcing. If this is the case, we can deduce that factors other than anthropogenic activity must play a role in the decadal transition of El Niño types. In this study, we examine multiple long-term instrumental SST datasets and apply cluster analysis to classify El Niño events. We show that there exists another extreme El Niño and CP El Niño epoch, around year 1900, with similar spatial and temporal evolution, dynamic processes, and climate impacts as those that occurred in the last 40 years. The results suggest a role of internal variability. Moreover, we found that both the two periods with increased extreme El Niño and CP El Niño events coincide with the positive phase of the AMO. Then, we investigate the influence of internal variability associated with the AMO on El Niño multidecadal modulation using outputs from the fifth and sixth phases of the Coupled Model Intercomparison Project (CMIP5 and CMIP6) and palaeoclimate proxies. Furthermore, we quantify the contribution of anthropogenic forcing and internal variability to the recently observed El Niño diversity based on a statistical model. Our results highlight that both internal variability associated with the AMO and anthropogenic forcing contribute to the changes in El Niño properties in recent decades.It is still unclear whether the frequent occurrence of extreme El Niño events and CP El Niño events in the last 40 years is part of natural variability31, Extended Reconstructed SST version 5 (ERSSTv5)32, and Kaplan Extended SST33 (see Methods). Based on the cluster analysis, El Niño events were classified into 4 physically meaningful clusters: (1) extreme, (2) EP, (3) CP, and (4) successive El Niño events . We focus on the extreme, EP, and CP types of El Niño events in this study. It is intriguing that almost all extreme and CP events occurred in the two periods. One is the period from the 1980s to the present, in which ~8 CP and 3 extreme events exist, and the other is the period 1870s to the 1900s, among which 3 extreme and 3 CP events consecutively occur  region during the El Niño development phase. It shows that during the onset phase, the zonal advective feedbacks are dominant for the extreme events in these two periods  during the preceding boreal winter and then propagates eastward with a rapid basin-wide development in the boreal spring. Later, a warm anomaly occurs in the far eastern Pacific during boreal spring and then propagates westward. The maximum intensity occurs around 120°W in December Fig. . Strong ods Fig. , and thi13; Supplementary Table The CP El Niño events in the two periods also show a similar evolution and feature initial warming in the western Pacific and eastward propagation from the western Pacific from early boreal summer to boreal winter Fig. . The onsp < 0.001). The boreal autumn air temperature anomaly patterns during CP El Niño events that occurred from 1875 to 1905 are also similar to those that occurred in the post-1980s . The result indicates that the regime shifts in the 1900s and 1980s are both significant at the 99% confidence level (Supplementary Table 39 (see Supplementary Discussion), and then we further divide the EP type El Niño events into moderate EP and extreme El Niño events by the criterion of DJF-averaged value of the normalized Niño3 index below or above a threshold value of 1.75 standard deviations (s.d.)  based on the Niño method41. Figure 41. The enhanced zonal advective feedback over the CP is conducive to triggering the development of El Niño over the western Pacific region42. We noted that both extreme and CP El Niño events are associated with an initial warm anomaly in the western Pacific, while EP El Niño events are not . A total of 29 of 43 CMIP5 and CMIP6 models (67.4%) produce an increased occurrence of CP and extreme El Niño for the AMO-positive state minus the AMO-negative state.We further analyzed the relationship between the AMO and El Niño diversity based on outputs from the fifth and sixth phases of the Coupled Model Intercomparison Project (CMIP5 and CMIP6). We use outputs from 20 CMIP5 and 23 CMIP6 pre-industrial control simulations over the last 300 years. The CMIP5 and CMIP6 models could reproduce the observed AMO characteristics, with similar spatial patterns in the North Atlantic Ocean  AMO index reconstruction47, and a 400-year (1617–2008) record of CP El Niño events reconstructed from ENSO-sensitive proxy records15. To focus on the decadal modulation of different types of El Niño events, we count the occurrences of extreme El Niño events (defined by a Niño-3.4 index >1.2 s.d.) and CP Niño events (identified following the pioneering work of ref. 15) over a 21-year sliding period. The 21-year sliding frequencies of extreme El Niño events and CP Niño events increased in the positive phase of the AMO  El Niño Niño3.4 index reconstructionAMO Fig.  and they45. Such a spatial pattern of the Pacific response implies that the AMO could induce anomalous high pressure over SNP  (b) the Extended Reconstructed Sea Surface Temperature (ERSST) version 5 global SST monthly dataset32 and (c) the Kaplan extended SST version 2 dataset33 for 1871–2017. Data uncertainties exist prior to 1950 due to the sparse coverage of instrumental observations across the equatorial Pacific54. To reduce the uncertainty in the three SST datasets, we used a “merged monthly mean SST dataset (merged SST hereafter) following the pioneering work of ref. 34”. The merged SST is defined as the arithmetic mean of the three monthly mean SST datasets. In addition to the “merged” SST dataset, we also used the HadISST1, ERSSTV5, and Kaplan SST datasets to examine the sensitivity of the results to different instrumental datasets  the Hadley Center Sea Ice and SST dataset version 1  with a 1° × 1° grid58. We use the difference between GODAS and SODA2.2.4 during the overlay period 1980–2008 to calibrate the mean state of GODAS. The land air temperature used is the HadCRUT559 gridded monthly surface temperature datasets. The long-term ship-observed sea level pressure (SLP) and marine cloud cover were used from the International Comprehensive Ocean–Atmosphere Data Set (ICOADS) release 360. The SLP field derived from the Hadley SLP (HadSLP2) dataset61 was also used.The surface winds and atmospheric circulation fields are derived by merging the NOAA-CIRES Twentieth Century Reanalysis (20CRv2c)20 and a 1200-year (800–2008) Atlantic Multidecadal Variability/Oscillation (AMV/AMO) Reconstructions based on 46 annual resolved terrestrial proxy records47. A 21-year running mean is applied to the AMV/AMO reconstruction index. The first and last 100 years are cut-offs to minimize edge effects introduced by the spectral filter. The overlay period of 1401–1900 is used. We define extreme El Niño as when the Niño 3.4 index is greater than 1.2 standard deviations. In addition to the multi-century record of extreme El Niño events reconstructed from the Niño-3.4 reconstruction, we also use 400 years (1601–2008) of CP El Niño events reconstructed from 27 seasonally resolved coral records15 to study the relationship between CP events and AMO. The period of 1601–1900 is used, and a total of 34 CP events are identified based on the pioneering work of ref. 12: 1618, 1620, 1641, 1652, 1657, 1667, 1672, 1677, 1682, 1688, 1693, 1718, 1730, 1733, 1759, 1769, 1775, 1778, 1779, 1781, 1790, 1799, 1801, 1808, 1816, 1832, 1840, 1850, 1853, 1854, 1873, 1884, 1885, and 1895 years.We also use multi-century palaeoclimate reconstructions to extend the observational record. The Niño-3.4 index and AMO index reconstructions used are from the NOAA/World Data Center for Paleoclimatology archive, including a 700-year (1300–2006) El Niño Niño-3.4 index reconstruction based on 2,222 tree-ring chronologies from both the tropics and mid-latitudes in both hemispheres63.We used all forcing (ALL) and single-forcing experiments forced separately by greenhouse gases (GHGs), natural forcing (NAT), and anthropogenic aerosols (AAs) from 27 CMIP6 models , here called the Niño-3.4 index. We denote the El Niño first developing year, the following year, and the year prior to El Niño as year 0, year 1, and year −1, respectively. We smooth the Niño-3.4 index with a 3-month running-mean filter. El Niño events are defined as occurring when the detrended October–February (ONDJF)-averaged Niño-3.4 index is greater than or equal to 0.6 °C. A total of 38 El Niño events are identified using the merged SST dataset from 1871 to 2017.34.We used a nonlinear K-means cluster analysis focused on the spatiotemporal evolution of El Niño events from the onset to the mature phase (from Nov-1 to Oct 0), which is depicted by SST anomalies averaged between 5°S and 5°N. The data used in the cluster analysis were 3-month running mean SST anomalies based on the original SST data, and to focus on the onset phase to mature phase, only SSTAs greater than 0.3 °C were used for the K-means cluster analysis, which is different from the pioneering work of ref. 39 to categorize El Niño events into EP and CP events and then extracted the extreme El Niño events  is the ocean water density; −1 K−1) is the specific heat of water; To compare the dominant dynamic processes of extreme and CP types of El Niño between the two periods, we conduct an ocean mixed layer heat budget analysis. The heat budget is computed according to the following equation:64. Pearson’s chi-square test statistic follows an asymptotic chi-square distribution with (r–1)Х(c–1) degrees of freedom when the row and column variables are independent. It is calculated asThe contingency table, also known as a cross-tabulation or a two-way table, is usually used to present categorical data in terms of frequency counts. We use Pearson’s chi‐square test to examine independence between the row and column variables in the contingency table65. This method is based on the principle that the internal variations among the ensemble runs are usually uncorrelated. Thus, after averaging over a large number of ensemble simulations, the uncorrelated internal variations among the ensemble runs were largely smoothed out65. To calculate the MMM, we averaged all the ensemble runs for each single-forcing simulation from all the models. The MMM was similar if we first average over the available ensemble runs for each model to derive the ensemble mean for each model and then average the ensemble mean over all the models with the same weight for each model. Then we calculate the GMSST time series, 65.Many previous studies used the global-mean SST (GMSST) from the multi-model ensemble mean (MMM) simulation to represent the externally forced signal. It is a preferred way to define the forced signal, which has the same temporal evolution over all grid boxes because it is determined by the external forcing seriesSince there is a good relationship between the background zonal SSTA gradient and the frequency of different types of El Niño occurrence, for example, frequent occurrence of extreme and CP El Niño events is observed in the period of 1875–1905, 1930–1949 and 1981–2017 coincide with the enhanced zonal SST gradient, while in the other period (from 1905 to 1930 and from 1950 to 1980), EP El Niño events are dominant with a weakened zonal SST gradient. Here, we attempt to estimate the relative contribution of EX- and IV-induced zonal gradients to the El Niño type changes in the last 40 years. We use the background zonal SSTA gradient as predictor N = 20 and used them to calculate the intercept We used the merged SST dataset shown in Fig. 66. We calculate the effective degrees of freedom (EDF) using the following equation:67 and use the above equation but without the weighting function (N - j)/N.Statistical significance tests were based on Student’s t test with reduced degrees of freedomWe use a bootstrap test to examine whether the number of extreme El Niño/CP El Niño events is statistically different in the positive phase of AMO and negative phase of AMO. We use Monte Carlo bootstrapping to estimate the probability density function (PDF) of the number of the 21-year sliding frequencies of extreme El Niño events and the number of the 21-year sliding frequencies of CP El Niño during positive and negative phases of the AMO. The resampling procedure was repeated 10 000 times. The 2.5 and 97.5% rankings from the probability distribution function indicate the 95% confidence level. The number of extreme El Niño increased in positive phase of AMO compared to negative phase of AMO is statistically significant above the 95% confidence level if distributions of number of extreme El Niño during positive phase of AMO are well-distinguished from that during the negative phase of AMO.Supplementary InformationPeer Review File


Threads of modern pedicle screws can vary greatly in design. It is difficult to assess which interplay of design features is particularly advantageous for screw anchorage. This study aims to increase the understanding of the anchorage behaviour between screw and cancellous bone. Pull-out tests of six pedicle screws in two sizes each were performed on three densities of biomechanical test material. More general screw characteristics were derived from the screw design and evaluated using the test data. Selected screws were tested on body donor material. Some screw characteristics, such as compacting, are well suited to compare the different thread designs of screws with tapered core. The combination of two characteristics, one representing bone compacting and one representing thread flank area, appears to be particularly advantageous for assessing anchorage behaviour. With an equation derived from these characteristics, the pull-out strength could be calculated very accurately (mean deviation 1%). Furthermore, findings are corroborated by tests on donor material. For screws with tapered core, the design demands for good anchorage against pull-out from cancellous bone change with material density. With sufficient bone quality, screws with a high compacting effect are advantageous, while with low bone density a high thread flank area also appears necessary for better screw anchorage. However, the choice of screw diameter and screw length strongly depends on the individual anatomy and surgical technique10. In addition, the bone quality is decisively influenced by the patient's state of health and age11. Thread design, on the other hand, can be adapted and is therefore the subject of research and development. Various design features of screws and their influence on anchorage have already been studied14. In some cases, screws have been manufactured specifically for the purpose of comparison, differing only in one design feature17. However, which combination of features provides the best anchorage is not yet fully understood. As a result, the screws used in clinical practice have very different thread designs.Screw diameter, screw length, screw design, and bone quality are often mentioned in the literature as the most important factors influencing pedicle screw anchorage23. Often this is conjecture, or there is little data available. There is a lack of comprehensive studies to further assess the relevance of these characteristics. Furthermore, it is assumed that the compacting of the bone together with the thread flank area is decisive for the anchorage of screws, but corresponding studies are lacking24. An analytical equation that takes into account the screw diameter, screw length, screw design and bone quality appears valuable for screw development and evaluation of the anchorage effect. Chapman et al.14 have proposed an equation for screw pull-out here. However, this has weaknesses as it does not include the effects of pre-drilling or tapping or the compaction effect of modern screws25.One approach is to look at more general screw characteristics, taking into account several design features. These may allow statements about the achievable anchoring effect. For example, screw characteristics such as flank overlap area, contact area, bone compacting or insertion torque have been suggestedThe aim of this work is to broaden the understanding of the anchorage behaviour between screws and cancellous bone. To this end, examinations are carried out in four steps. First, six types of pedicle screws, each in two sizes, are tested in pull-out tests on three densities of synthetic foam, and the measurement results are compared. Secondly, it is examined whether more general screw characteristics are suitable for assessing screw anchorage. Thirdly, it is analytically evaluated whether the combination of two parameters, one describing the compacting of bone and one describing the thread flank area, allows statements about screw anchorage. For this purpose, an equation for calculating the pull-out strength from these parameters is established. The results are compared with the measurement results and Chapman's equation. Finally, the findings obtained on synthetic material are verified on a more complex material. For this purpose, pull-out experiments are performed on cancellous bone from human donor material in order to classify the results quantitatively.Six screw types were selected, each with two different outer diameters. All of them were approved pedicle screws in clinical use, with the exception of the screw type 5, which was a prototype . The screws were made of titanium alloy and 45 mm long. The thread region of all screws is shown in Fig. 26 in grades PCF 15 (0.24 g/cm3), PCF 10 (0.16 g/cm3), and PCF 5 (0.08 g/cm3) was used as homogeneous test material. Foams with apparent densities of 0.24 g/cm3 were used as a model for normal, 0.16 g/cm3 for osteoporotic and 0.08 g/cm3 for severely osteoporotic bone5. The densities of each group may vary slightly (up to ± 10% according to the manufacturer). To obtain reproducible results, recommendations for sample preparation were followed27. The apparent density was determined for each foam block according to ASTM D162228. Foam blocks with as equal a density as possible were selected. Next to this, the blocks were divided so that each screw was tested in each foam block. In addition, the foam blocks were pre-drilled perpendicular to the surface to a diameter of 2.5 mm up to a length of 28 mm.Polyurethane foam  according to ASTM F183929, cf. Fig. All screws were inserted perpendicularly into the foam blocks by machine on a self-developed screw-in test rig based on ASTM F543Subsequently, the screws were pulled out of the foam as per ASTM F543. Accordingly, a spacing of the screws in the foam of at least five times the diameter was maintained. In addition, the opening of the hold-down was at least five times the screws’ diameter. Finally, the screws were pulled out axially and perpendicular to the foam specimen at a speed of 5 mm/min. The receptacle for the screw head caused the application of pure tensile forces and prevented bending moments, cf. Fig. outer diameter31. The flank overlap areao, mean core diameter Dc, screw length L, and screw pitch p18. The contact surfacecompacting boned as displaced bone volume when a screw was inserted24. The displaced volume was obtained from virtual 3D models of the screws and corresponded to the volume of the screw part located in the bone material minus the volume of the pilot hole. Furthermore, the displaced diameter24. Moreover, the insertion torque21. In addition, the product of outer diameter and insertion torqueproduct of outer diameter and compacting24.Eight screw characteristics (obliquely highlighted) were identified and tested for a linear relationship with the measured pull-out strength. Here, the 14 proposed an equation for calculating the pull-out strength of cancellous bone screws asDuring further analysis, the influence of compacting together with the thread flank area on the pull-out strength was evaluated analytically. Chapman et al.s as the shear length). Whereby the so-called thread shape factor TSF considers the thread design over the thread depth This takes into account the shear strength In this study, the Chapman equation was built upon. The correction factor First, the ideal correction factor s were determined experimentally , institutional approval for the use of the post-mortem tissues of human body donors was obtained. The authors declare that all experiments were performed according to the ethical principles of the Declaration of Helsinki.Twelve fresh-frozen human vertebrae (th11 and th12) from six body donors  were harvested, cf. Table The vertebral bodies were embedded in a ring with a polyurethane casting resin . Two screw types (ST2_7 and ST3_6.2) were examined and each tested on one vertebral body. The test was performed analogously to the tests on synthetic foam. One screw was screwed into the vertebral body laterally on the left, the other laterally on the right. Sufficient space was left between the screws to rule out any interference. Careful planning of the drill holes and control of the screw-in path during pre-drilling ensured that the screws were inserted in a purely cancellous manner. This was followed by the axial pull-out, cf. Fig. 32. Linear regression analyses were carried out to examine the influence of different screw characteristics on the pull-out strength and the relationship between insertion torque and pull-out strength. For correlations with two parameters, a 3D regression was performed. The coefficient of determination R2 was determined to assess the correlation. In all statistical analysies a value p < 0.05 was considered statistically significant.Statistical analyses were performed with SPSS 24.0 . The Shapiro–Wilk test was used to check the series of measurements for normal distribution. Differences in the means of the synthetic foam specimens were analysed with the two-tailed t-test for normally distributed data, otherwise the Mann–Whitney test was used. The measurement data from the experiments with donor bones were tested with the two-tailed paired t-test. In addition, Cohen's effect size d was determined p = 0.15 (PCF 15), p = 0.038 (PCF 10), p < 0.001 (PCF 5)] and the pull-out strength  were determined for the screws ST2_7 and ST3_6.2.The measured values  are listed in Table 2 > 0.7 in italics and non-significant data highlighted in bold. The corresponding diagrams with data points of the mean values for screws with tapered core are shown in Fig. Values for the previously defined screw characteristics can be found in Table Figure 2 for the respective parameter combinations of An example of a 3D regression is shown in Fig. from Eq. , leadingBased on this, the pull-out strength g to Eq. .Table range: −  to 33%. 2, cf. Table p = 0.18). For the pull-out strength 148 N (SD 50 N) was measured for ST2_7 and 175 N (SD 67 N) for ST3_6.2. The mean value difference in pull-out strength was 26 N (SD 35 N) and was statistically significant p = 0.04 . The Cohen's d value was determined to be 0.75, corresponding to a medium to large effect. In direct comparison, the relative mean deviation of the pull-out strength for ST3_6.2 is 12% higher than for ST2_7. A linear correlation of the pull-out strength to the BMD was found for the screw ST2_7  and a high linear correlation for ST3_6.2 , cf. Figure 2 = 0.92, p < 0.001) and ST3_6.2 .The DEXA examination resulted in T-scores for one osteopenic (T-score: − 1.9) and five osteoporotic (T-score: − 3.1 to − 4.7) body donor samples. Bone mineral density ranged from 599 to 977 mg/cmIn a comprehensive study, the anchoring effect of screws in the cancellous bone was analysed. It has been shown that some screw characteristics derived from the screw design are suitable to assess the anchorage quality against pull-out from artificial cancellous bone. In fact, it seems more useful to consider two parameters together, one describing the compacting and one describing the thread flank area. To the authors' knowledge, this is the first time that the effects of two parameters in combination on the anchorage behaviour of pedicle screws have been investigated, taking into account different material densities. For screws with tapered core, the parameters of displaced diameter and relative thread flank area proved to be the most suitable. An equation derived from these parameters provides very good predictions for the expected pull-out strength and is an improvement on the Chapman equation. Furthermore, results could be verified by tests on donor material. These findings will be discussed in detail below.34. It is still unknown which combination of features leads to the best anchorage. The interaction of design features has hardly been considered so far. In this study, the characteristics of screws resulting from different combinations of design features were studied. These characteristics were expressed as quantitative values. As a result, comparisons are possible even for screws that differ in more than one design feature. This approach seems advantageous as different screws can be compared with each other as their thread design is described by more general characteristics.Each design feature can have an influence on screw anchorage31. In the current study, it is found for screws with tapered core that higher pull-out strengths are achieved with a larger outer diameter for one type of screw, cf. Table It is often assumed that a better anchorage is achieved with a larger outer diameter36. Therefore, these characteristics are not sufficient as stand-alone parameters for assessing screw anchorage. Krenn et al.18 who introduced FOA come to the same conclusion for the characteristic FOA.For screws with tapered core, good correlations of flank overlap area FOA and contact surface to pull-out strength were found, cf. Fig. 24. For screws with tapered core, very good correlations to pull-out strength are achieved over these three characteristics, especially at higher foam densities, cf. Table 23. For the first time, a quantitative comparison of the compacting for a larger number of screws has been considered. This characteristic appears useful for understanding the anchoring mechanisms. However, the degree of compacting cannot be increased arbitrarily. While at higher densities the degree of compacting has a substantial influence, it seems that at lower densities FOA becomes more important, cf. Fig. 25 come to a similar conclusion when they state that pull-out strength is the result of a number of varying parameters.The screw hole preparation influences how much material is compacted between the thread flanks. The characteristics displaced diameter 38. This view is controversial in the literature and has been discussed in detail elsewhere24. However, the insertion torque is determined during surgery and is therefore not a characteristic that results directly from the thread design.The relevance of compacting and flank area can also be observed for screws with the same outer diameter. ST3_7 has higher compacting and a larger flank area than ST1_7 and ST2_7, which is also reflected in a higher pull-out strength for all three material densities. ST1_6 and ST2_6 have similar compacting properties. However, ST1_6 has a larger flank area, which is also reflected in a higher pull-out strength. As a further result, a very good correlation between pull-out strength and insertion torque, as well as to the term 40. Asnis et al.39 mentioned that this effect is more pronounced at low densities. In the current study, different compacting parameters are considered, but it seems that compacting is best represented by the displaced diameter.In an analytical evaluation, the influence of the screw design on the screw hold was mapped via two parameters as a correction factor 25. In Table 39. In addition, the difference between the ideal correction factor 25 presented a complex integral approach for the calculation of the pull-out strength from the screw design. They achieved a mean deviation of − 5% (range: − 10 to − 2%). For this, they had determined the shear strength of the synthetic test material and used only one density. It stands to reason that with the approach presented, the pull-out strengths could be calculated even more accurately if only one density is considered. This can be seen from the good correlations of the individual densities, cf. Fig. For screws with tapered core, an equation for calculating the pull-out strength from the parameters displaced diameter and relative flank area was presented, cf. Equation  with Eq.In any case, it was not the intention of this study to derive a new universal equation for calculating the pull-out strength of pedicle screws. This would require further research to map, among other things, the compacting for different screw hole preparation techniques and the relationship with changing material density. The aim was rather to verify the assumption that the anchoring effect against pull-out is determined by the compacting and the thread flank area. In Eq. , the par40. However, a significantly higher pull-out strength in the donor material was demonstrated for ST3_6.2, as had already been done for synthetic foam. Consequently, the screw characteristics of ST3_6.2 result in a stronger screw-bone bond compared to ST2_7. The screw design had an effect on the anchorage. Presumably, the higher compacting effect of this screw leads to better anchorage. Possibly, this is further enhanced in low bone quality due to the larger relative thread flank area. In any case, this is an interesting result and should be further investigated in subsequent studies with more screws and specimens. Furthermore, as in other studies with donor material, a correlation between BMD and pull-out strength as well as between insertion torque and pull-out strength  also appears necessary for better screw anchorage.Supplementary Information.


The in vitro digestion method simulated the stages of digestion that occur in the mouth, stomach and small intestine under optimal controlled conditions . At each stage of digestion, the survival rate of probiotic bacteria was determined using the plate-deep method. As expected, in the oral stage, there was no significant reduction in the viability of the probiotic bacteria in any ice cream group compared to their content before digestion. In the stomach stage, Bifidobacterium animalis ssp. lactis BB-12 strain had the highest viable counts (8.48 log cfu g−1) among the control samples. Furthermore, a 4% addition of inulin to ice cream with Bifidobacterium BB-12 increased gastric juice tolerance and limited strain reduction by only 16.7% compared to the number of bacterial cells before digestion. Regarding ice cream samples with Bifidobacterium BB-12, replacing part of the inulin with apple fiber resulted in increased survival at the stomach stage and a low reduction in the bacterial population of only 15.6% compared to samples before digestion. At the stomach stage, the positive effect of the addition of inulin and apple fiber was also demonstrated for ice cream samples with Lacticaseibacilluscasei 431 (9.47 log cfu g−1), Lactobacillus acidophilus LA-5 (8.06 log cfu g−1) and Lacticaseibacillus paracasei L-26 (5.79 log cfu g−1). This study showed the highest sensitivity to simulated gastric stress for ice cream samples with Lacticaseibacillusrhamnosus (4.54 log cfu g−1). Our study confirmed that the 4% addition of inulin to ice cream increases the survival rate of L. casei and Bifidobacterium BB-12 in simulated intestinal juice with bile by 0.87 and 2.26 log cfu g−1, respectively. The highest viable count in the small intestine stage was observed in ice cream with L. acidophilus. The addition of inulin increased the survival of L. rhamnosus by 10.8% and Bifidobacterium BB-12 by about 22% under conditions of simulated in vitro digestion compared to their control samples. The survival rates of L. casei and L. paracasei were also highly affected by the 4% addition of apple fiber, where the increase under gastrointestinal passage conditions was determined to range from 7.86–11.26% compared to their control counterparts. In comparison, the lowest survival rate was found in the control ice cream with L. rhamnosus (47.40%). In our study at the intestinal stage, only five ice cream groups: a sample with 4% inulin and L. acidophilus, a control sample with Bifidobacterium BB12, a sample with 2.5% inulin and 1.5% apple fiber with Bifidobacterium BB12, a control sample with L. rhamnosus, a sample with 4% fiber and L. rhamnosus reported bacterial cell counts below 6 log cfu g−1 but higher than 5 log cfu g−1. However, in the remaining ice cream groups, viable counts of bacterial cells ranged from 6.11 to 8.88 log cfu g−1, ensuring a therapeutic effect. Studies have clearly indicated that sheep milk ice cream could provide a suitable matrix for the delivery of probiotics and prebiotics and contribute to intestinal homeostasis. The obtained results have an applicative character and may play an essential role in developing new functional sheep milk ice cream.We conducted a study to determine the survival of bacterial cells under in vitro digestion. For this purpose, ice cream mixes were prepared: control, with 4% inulin, 2.5% inulin and 1.5% apple fiber and 4% apple fiber. Each inoculum (pH = 4.60 ± 0.05), containing 9 log cfu g In the recent decade, it has become common practice to integrate bioactive ingredients into food products to promote health and personal well-being . Ice cre−1 [2+ has been found to increase the yield and biological activity of exopolysaccharides [Sheep milk is an excellent raw material for producing probiotic ice cream. Sheep milk differs from cow milk in chemical composition, including higher protein and fat content, and physicochemical properties . High pr−1 . As an i−1 . The addcharides ,7,8, whiProbiotics are defined as live microorganisms that, when provided in adequate amounts, are beneficial to the host’s health ,10. ProbL. rhamnosus and Bifidobacterium lactis combined with prebiotic inulin enriched with oligofructose provide anticancer effects [L. acidophilus and L. casei also exhibit immunoenhancing properties by increasing the phagocytic activity of granulocytes, the activity of immunoglobulin-secreting cells in the blood, and the excretion of cytokines in lymphocytes [Bifidobacterium spp. and L. acidophilus reduce blood cholesterol by causing the deconjugation of bile salts, binding cholesterol to bacterial cell walls and contributing to the reduction of cholesterol biosynthesis [Bifidobacterium and Lactobacillus have been proven to reduce obesity and insulin resistance by regulating metabolism. The prevention of gastrointestinal infections is a consequence of probiotics’ production of substances with antimicrobial properties, such as organic acids, mainly lactic and acetic acids, and through the stimulation of immune processes in the host. To induce a beneficial probiotic effect, the minimum therapeutic dose of a probiotic should be 106–109 CFU.Probiotic bacteria have the ability to spread from the gastrointestinal tract to extraintestinal sites through dendritic cells. These cells can penetrate the epithelium and absorb bacteria directly from the intestinal lumen. After entering dendritic cells or macrophages, probiotic bacteria can be transported to other locations by circulating immune cells in the bloodstream. Bacterial adhesion to the host surface is a crucial aspect of host colonization, as it prevents the mechanical clearance of pathogens . Further effects . L. acidphocytes . The proynthesis ,24. FurtDue to growing consumer interest, products containing cultures of live probiotic microorganisms have become an essential commercial commodity, coming in various forms: tablets, sprays, liquids, suspensions, capsules, powder sachets, granules and in food . Probiot−3 upH/min for milk fermented by L. bulgaricus, L. acidophilus, L. rhamnosus and B. animalis ssp. lactis BB-12 with co-cultures of S. thermophilus. When 4% inulin was added to the milk, there was an increase in the time to reach Vmax, which ranged from 17.0 to 19.0 × 10−3 upH/min [Optimal conditions for the growth of probiotic bacteria include a temperature of 30–40 °C and a pH of 5.5–6.2, but they can grow at a temperature of 2–53 °C . The lit upH/min .Lactobacillus and Bifidobacterium [Probiotic bacteria cells are destroyed during freezing and aeration of the ice cream mixes and in storage; however, many researchers have shown that probiotic cultures have better viability in frozen products than in fermented milk ,34,35. Tacterium ,37. The acterium ,39,40.Prebiotics are substrates that pass through the esophagus and stomach, reaching the intestines in undigested form and providing an abundant energy source for intestinal microflora ,41,42. RLacticaseibacillus casei 431, Lactobacillus acidophilus LA-5, Bifidobacterium animalis ssp. lactis BB-12, Lacticaseibacillus paracasei L-26 and Lacticaseibacillus rhamnosus, by simulated in vitro digestion in ice cream manufactured from sheep milk, using different doses of apple fiber and inulin.Sheep milk ice cream combines the health-promoting properties of sheep milk with the beneficial effects of fiber and probiotics on human health. Studies on synbiotic sheep milk ice cream are important for ice cream manufacturers, consumers and nutritionists. These studies disprove of the myth of ice cream as a provider of empty calories consisting of sugar. Sheep milk ice cream provides a matrix rich in protein, fat and vitamins, further protecting probiotic cells. Therefore, it is crucial to show whether probiotic monocultures applied in the dairy industry can survive under simulated gastrointestinal conditions and induce a beneficial probiotic effect. The study aimed to evaluate the survival of five strains of probiotic bacteria, namely 2HPO4; 141.96 g/mol; Chempur, Piekary Śląskie, Poland), di-potassium hydrogen phosphate , sodium chloride pure p.a. ≥99.9% , hydrochloric acid 12 mol , sodium hydroxide 1 mol . All of the reagents used were of analytical reagent grade.Enzymes and reagents: heat-stable α-amylase , mucin from the porcine stomach , pepsin from the porcine gastric mucosa , porcine bile extract , pancreatin from the porcine pancreas , di-sodium hydrogen phosphate anhydrous pure p.a. ≥99.0%  were purchased from Chr. Hansen . These starter cultures are widely used by the dairy industry to produce probiotic fermented milk and are not genetically modified.Materials used in the manufacture of ice cream mixtures: raw sheep milk was purchased from the farm “Owcza Zagroda” ; inulin ; apple fiber ; and white sugar . Five types of commercial freeze-dried starter cultures for direct vat set (Sheep milk (89%) with sugar (11%);Sheep milk (85%), sugar (11%), inulin (4%);Sheep milk (85%), sugar (11%), inulin (1.5%), apple fiber (2.5%);Sheep milk (85%), sugar (11%), apple fiber (4%).For each probiotic bacteria, 4 groups of mixtures with additives were prepared:−1 (w/w), were activated at 40 °C until the pH was 4.60 ± 0.10. Bacteria inoculums were approximately 9 log CFU bacteria g−1. The inoculum was added to ice cream mixes in the amount of 5% (w/w). Prior to adding inoculum, each mixture was homogenized with a homogenizer  and then pasteurized at 85 °C, 1 min. After the heat treatment, the mixtures were cooled to 37 °C and fermented in an incubator  at 37 °C for ten hours, then cooled to 5 °C and conditioned at this temperature for 12 h. The ice cream mixtures were frozen in a DeLux 48,816 freezer  for 40–50 min with freezing temperatures down to −22 °C. The ice cream was packed into 100 mL plastic cups and stored at −22 °C for seven days. The experiment was repeated three times. The ice cream was coded according to the probiotic bacteria type and the different fiber and inulin doses  or NaOH (1 mol/L) and incubated in a shaking water bath at 37 °C and 90 rpm for 10 min.The in vitro digestion model was initiated from the oral stage. 50 mL of each sample was transferred to a 100 mL dark glass bottle and then mixed with 5 mL of salivary enzyme solution. The salivary solution was obtained by mixing and dissolving 2.38 g of NaThe stomach stage was initiated by adding 13.08 mg of pepsin to the sample after the oral stage, and the pH was reduced to 2.0 ± 0.20 by adding HCl (12 mol/L). The sample with digestive contents was placed in a shaking water bath for 2 h at 37 °C and 90 rpm.To obtain the intestinal fraction, the oral and gastric contents were mixed with 5 mL pancreatin (4 g/L) and bile salt (25 g/L) at a pH value of 7.00 ± 0.20 (HCl 12 mol/L or NaOH 1 mol/L) and incubation was continued for another 2 h .The survival rate (%) was determined in terms of viable colony counts of probiotic bacteria remaining in the intestinal content relative to the non-digested sample, according to Equation (1):L. paracasei L-26, L. casei 431, L. acidophilus LA-5, L. rhamnosus and Bifidobacterium BB-12 were determined in ice cream before digestion and at each stage of simulated in vitro digestion  after seven days of storage at −22 °C. According to the method of Znamirowska et al. [−1 and 8 log cfu g−1. Inoculation was performed by the plate-deep method using MRS agar . A vacuum desiccator and GENbox anaer  were used to maintain anaerobic conditions, while an Anaer indicator  was used to monitor anaerobic conditions. The cultured probiotic colonies were counted using a colony counter . The result was presented as log cfu g−1.The survival rates of probiotic strains: a et al. , 10 g ofp < 0.05).From the obtained results, the mean and standard deviation were calculated using Statistica v. 13.1 . One-, two-, and three-way ANOVA were performed. The significance of differences between the mean values was verified with the Tukey test , while the lowest number was determined in ice cream with L. acidophilus . The number of probiotic bacteria cells in all tested ice cream groups before digestion exceeded 6 log CFU g−1, which qualifies this product as probiotic ice cream and initially guarantees a therapeutic effect. However, only the number of cells determined at the small intestine stage will accurately indicate probiotic activity.Before digestion, the highest number of bacterial cells was determined in ice cream with During the freezing process, specific changes occur in ice cream manufacturing that cause the loss of metabolic characteristics due to changes in osmotic pressure in the cells. The ice crystals that form can mechanically damage the cell walls of probiotic bacteria. Adverse factors also include the effects of oxygen during the aeration of the mixture or during storage and high redox potential values . Howeverp = 0.000), the interactions between bacterial type and inulin addition (p = 0.0003), and the interactions between bacterial type and apple fiber addition (p = 0.0001)  . It was The results in Bifidobacterium BB-12 and the CLC samples with L. casei 431 were found to be the most susceptible to the acid stress that occurs during gastric passage, with population reductions of 73.93% and 65.52% compared to the number of viable cells before digestion. Slightly smaller population reductions in the stomach (43.59%) were found in the CLR control sample with L. rhamnosus. Probiotics must survive in the stomach’s acidic environment if they reach the small intestine and colonize the host. Lactobacillus species are thought to be intrinsically acid resistant [Bifidobacterium BB-12, where a population reduction of only 26.0% was found compared to the count before digestion. Furthermore, it was found that the addition of inulin to IBB12 ice cream increased gastric juice tolerance and reduced strain reduction to 16.7% compared to the number of bacterial cells before digestion. For IFBB12 samples with Bifidobacterium BB-12, replacing part of the inulin with apple fiber resulted in increased survival in the gastric stage and a poor reduction in the bacterial population of only 15.6% compared to the samples before digestion. However, the complete replacement of inulin with apple fiber in the IFBB12 ice cream resulted in a population reduction of 22.4%, but the survival rate was still 3.6% higher than in the CBB12 control sample. The obtained results indicate that the addition of these fibers to ice cream with Bifidobacterium BB-12 increases the survival of this strain in model gastric juice.Among the control samples, the CBB12 samples with esistant . Althougesistant ,64. Hencesistant . The resesistant . The enzesistant . It was L. casei, L. acidophilus and L. paracasei. The best survival of probiotics in the stomach was observed for ice cream samples with inulin partially replaced by IFLC apple fiber . This study demonstrated the highest sensitivity to simulated gastric stress for ice cream samples with L. rhamnosus. At the gastric stage, there was a reduction of 56.4% of the population in the CLR ice cream compared to the cell counts before digestion. Adding fibers to the samples with this strain resulted in an even higher 1–3% population reduction at the stomach stage compared to the CLR control sample. In the FLC, FLA, FBB12 and FLP ice cream samples, the 4% addition of apple fiber (without inulin) was found to increase bacterial survival more effectively than the presence of a combination of inulin (2.5%) and apple fiber (1.5%) in the gastric juice.In the gastric stage, the positive effect of the addition of inulin and apple fiber was also demonstrated for ice cream samples fermented with L. casei in simulated gastric juice. A faster reduction in cell counts (from 10.79 to 5.48 log10 cfu/mL) was observed for ice cream samples with free probiotics compared to ice cream, where probiotics were encapsulated (10.72 to 7.65 log10 cfu/mL). Other studies indicate that the addition of inulin to microencapsulated probiotics provides protection upon passage through the gastrointestinal tract [Numerous studies confirm the improved survival rate of microencapsulated probiotics under simulated in vitro digestion ,70 and rL. rhamnosus GG.The intestinal juice is secreted at a rate of 0.7 L per day. Its pH is around 8 and the concentration of mineral salts is about 0.5%. The factors determining the survival of bacteria in this section of the digestive tract are bile and the presence of enzymes. The second barrier to the survival of lactic fermentation bacteria in the gastrointestinal tract is bile acids produced in the liver from cholesterol and secreted into the duodenum. The concentration of bile acid salts fluctuates between 0.15% and 0.5%. Bile secreted into the digestive tract plays an important role in emulsifying lipids, especially since the fat content in the ice cream evaluated ranged from 6.2% to 6.4%. Furthermore, bile has the ability to affect phospholipids and cell membrane proteins and thus disrupt the cellular homeostasis of microorganisms ,74. BileBifidobacterium BB-12 due to a reduction in survival from 12.7–31.0% compared to the high number of cells determined in the stomach. It should be mentioned that Ruas-Madiedo et al. [Bifidobacterium in the presence of bile and explained that EPS probably poses a defense mechanism against this toxic compound. Ruiz et al. [B. animalis IPLA 4549 and its mutant with acquired bile resistance, B. animalis 4549dOx. According to these authors’ studies, bile adaptation caused B. animalis 4549dOx to decrease membrane fluidity and protein: phospholipid ratio, as well as alter the fatty acid composition of the cell. Unfortunately, our study does not confirm the high tolerance of the Bifidobacterium strain BB-12 to bile.In our study, the highest sensitivity to simulated intestinal stress was found for o et al.  detectedz et al.  investigL. casei, which was lower by 6.1–21.9% compared to the number of bacteria determined in the stomach . Presumably, the whey proteins in sheep milk contributed to better tolerance of L. acidophilus to the simulated digestive conditions of the gastrointestinal tract. In the study by de Figueiredo Furtado et al. [According to other authors’ studies, the survival rate of probiotic strains gradually decreased during in vitro passage through the stomach and small intestine ,80. In oo et al. , the phyL. acidophilus, the addition of inulin and apple fiber resulted in a 11.1–31.4% reduction in the survival of this strain compared to the CLA sample. Meanwhile, the lowest survival rate was found in the CLR control ice cream with L. rhamnosus. As shown by the results in L. rhamnosus by 10.8% compared to the CLR control sample under simulated in vitro digestion conditions. An even higher survival rate (about 22%) after inulin addition compared to the control sample was found for the Bifidobacterium BB-12 strain. This is consistent with the findings of Tarifa et al. [L. casei and L. rhamnosus in the absence of inulin significantly decreased, between 60% and 90%, after passage through the gastrointestinal tract, showing a 62.2% reduction in the Lactobacillus population in simulated in vitro digestion. Furthermore, Hu et al. [Lactobacillus population in simulated in vitro digestion. In contrast, the addition of only apple fiber improved the survival of Bifidobacterium BB-12 under simulated in vitro digestion by only about 6% compared to the control. Similarly, the survival rate of L. casei, L. paracasei, and L. acidophilus was also highly affected by the 4% addition of apple fiber, where its increase under gastrointestinal passage conditions was determined to range from 5.7–11.2% compared to their control counterparts. In apple fiber, soluble pectin represents 40% of the content, and about half is insoluble cellulose [In ice cream with a et al. , where tu et al.  showed aellulose . Pectin ellulose , which cellulose .L. rhamnosus that pectin cross-links with Ca2+ to protect probiotic cells from acid inactivation. Bifidobacterium ssp. show poor pectin degradation but are adapted to degrade side chains from arabinate and galactan [L. rhamnosus in gastric environments at a very low pH [Lactobacillus acidophilus showed less reduction (1.51 log cycles) than unencapsulated cells (3.54 log cycles) when incubated in gastric and intestinal juices. Additionally, a study by Tarifa et al. [L. casei and L. rhamnosus coated with pectin and inulin could improve viability under gastrointestinal conditions compared to free cells. At the end of the sequence, the survival rate of L. casei 393 was 51.00%, and the survival rate for L. rhamnosus was 61.00%.Krasaekoopt and Bhandari  also fougalactan . In a suy low pH . Gebara y low pH  describea et al.  showed t−1, but higher than 5 log cfu g−1. In the remaining ice cream groups, the number of viable bacterial cells ranged from 6.11 to 8.88 log cfu g−1, which qualifies them as probiotic ice cream and guarantees a therapeutic effect.In our study, in the small intestine stage, only five ice cream groups  had bacterial cell counts below 6 log cfu gThe three-way analysis of variance indicated that the survival rate and tolerance to conditions in the small intestine were significantly influenced by all the factors analyzed  and their interactions.The gastrointestinal tract passage might not be sufficient for good intestinal colonization . Since tLactobacillus ssp. can be divided into protein, non-protein, environmental, aggregation capacity and hydrophobicity. Factors such as bile salts, low pH, digestive enzymes or oxidative and osmotic stress affect the cell wall properties of lactobacilli and, thus, their adhesion abilities. Tuomola et al. [L. acidophilus LA-1 adhesion, indicating that the outer protein layer of the cell wall plays an essential role in cell adhesion. Moreover, Lim and Ahn [Lactobacillus strains, showed that L. plantarum GK81, L. acidophilus GK20, and L. paracasei GK74 strains, after incubation with pepsin, protease and trypsin, showed significantly lower adhesion to Caco-2 cells . It was also observed that the adhesion of probiotic strains is related to the growth phase of the microorganisms and that cells in the logarithmic growth phase adhere better than cells in the stationary phase [Adhesion is a process that allows microorganisms to adhere to other cells or surfaces . Structua et al.  showed t and Ahn , who stury phase .Additionally, for probiotic bacteria to benefit the host, they need to reach a sufficient mass. Therefore, the ability to aggregate is desirable among probiotic strains . The forThe popularity of frozen desserts continues to grow with new varieties and flavors. Food manufacturers can provide consumers with healthier probiotic- and prebiotic-providing frozen dessert options. An additional nutritional benefit would be for food manufacturers to provide a product that qualifies as probiotic ice cream and pre-guarantees a therapeutic effect, similar to the probiotic strains provided with drugs and food supplements. Sheep milk functional ice cream is a food product that contains valuable bioactive ingredients that offer health benefits beyond their nutritional value. The bioactive ingredients are synbiotics, a combination of probiotics and prebiotics, which are generally important for gut health.−1, demonstrating a good survival rate.The conducted studies clearly indicate that sheep milk ice cream could provide a suitable matrix for the delivery of probiotics from commercial dairy cultures and prebiotics and could contribute to intestinal homeostasis. The results indicated that in the small intestinal phase, all groups had a higher probiotic content than 5 log cfu gEvaluating the effect of fiber addition on the survival of probiotic bacterial strains in the gastrointestinal tract would be helpful in developing new and innovative products with enhanced health properties tailored to the needs of the intestinal ecosystem.


IgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI  suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown.We sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production.Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR.The compounds in Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2–20 µg/ml (p < 0.05–0.001 vs. untreated cells) with an IC50 value of 3.43 μg/ml. There was no cytotoxicity at any tested concentration. Formononetin significantly decreased XBP-1, and IgE-heavy chain gene expression compared with untreated cells (p < 0.001).A single compound identified as formononetin was isolated from Formononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases. There has been a steady increase in the prevalence of allergic diseases around the world. Allergy is now the 6th leading cause of chronic illnesses in the United States, with more than 50 million people suffering from the disease yearly . Globall+ B cells leading to the decrease in the number of these cells, which, in turn, leads to a decrease in the production of serum IgE. Suplatast tosilate inhibits the production of IL-4 and IL-5 in Th2 cells, which causes a decrease in the synthesis of IgE, It has been used in the treatment of allergic asthma , Sophorae flavescens (SF), and Glycyrrhiza uralensis (GU). ASHMI is effective in the treatment of Allergic Asthma, with clinical trials demonstrating acceptable levels of safety and tolerability , Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals . 2-Mercaptoethanol (2-ME), sodium pyruvate,  (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich . Acetonitrile, dichloromethane, ethylacetate, methanol, and formic acid were purchased from Fisher Scientific .ASHMI product is a dried aqueous extract of SF, GU, and GL, in capsule form . ASHMI aSF provided by Sino-lion Pharmaceutical Company (P.R. China), was dissolved into 4 L water, and mixed with 3 L of CH2Cl2 in liquid-liquid extractor. 24 h later, the non-polar fraction was collected and dried on a rotary evaporator under low pressure. Methanol (300 ml) was used to re-dissolve the dried residues. The mixture was centrifuged at 2,500 rpm for 30 min and further separated with an AutoPurification system coupled with UV/Vis Detector . Four fractions were collected based on the major peaks presented in the analytic HPLC chromatogram data.Dichloromethane extract of SF was performed utilizing liquid-liquid extraction method. Briefly, 50 g of Compounds characterization was done using a Waters 2695 HPLC system  coupled to a Waters Micromass LCT Premier mass spectrometer. Samples were separated on an analytical Column: Zorbax® SB C18   with 0.1% formic acid as mobile phase A and acetonitrile with 0.1% formic acid as mobile phase B. A gradient program was used as follows: 0–75 min, linear change from A–B  to A–B . A 15 min equilibration time was used before each HPLC run. The flow rate was set at 1 ml/min. The UV spectra were recorded between 200 and 460 nm. The detection wavelength was set at 254 nm, as previously described  in both electrospray (ESI) positive and negative mode. 10 μl of sample was analyzed under the same gradient condition described above. The parameters were set as: Capillary Voltage: 3,200 v; Cone Voltage: 15 v; Aperture I: 25 v; Desolvation Temperature: 300°C; Source Temperature: 110°C; Desolvation gas: 500 L/h; Nebulizing gas: 40 L/h; Ionization mode: Electrospray; Positive Ion Acquisition Range: m/z 50–1,000. The results were collected and analyzed by Empower and Masslynx software . In some experiments, U266 cells were treated with active compound for 3 days. Cells were then subjected to western or qPCR analyses.The human U266 myeloma cell line, purchased from ATCC  is widely used in the allergy research to screen the effects of compounds on B cell IgE production  on day 0, supernatants were harvested after 3 days culture, and IgG levels were determined by using an ELISA Kit .ARH-77 cell line, purchased from ATCC  to screen the effects of compound formononetin on B cell IgG production . Cells wCell viability was evaluated using a trypan blue exclusion as previously described. Briefly, a 10 µl of cells suspension from each culture was mixed with equal volume of trypan blue dye . The mixCell cytotoxicity was evaluated using cell counting kit-8 according to manufacturer's instruction, and previously published including ours , 29. We 6 cells/ml) were co-incubated with formononetin (10 µg/ml) at 37°C under 5% CO2 for 3 days. Cells were then harvested, and total RNA was isolated using Trizol reagent  following the manufacturer's instructions. The RNA concentrations were then quantified by triplicate optical density (OD) readings . The reverse transcription was performed to get the cDNA using RevertAid RT Reverse Transcription Kit  as described by the manufacturer's instructions. Pre-chill sterile, thin-walled tubes and reaction tubes on ice, pre-chill nuclease-free water. Combine the RNA (up to 1 µg) and the cDNA primer in nuclease-free water for a final volume of 5 µl per RT reaction on ice. Place the tube into preheated 70°C heat block for 5 min immediately chill the tube on ice for 5 min spin each tube to collect all condensate. Prepare the reverse transcription reaction mix ImProm-II™ 5X Reaction Buffer, MgCl2, dNTP, Recombinant RNasin® Ribonuclease Inhibitor, ImProm-II™ Reverse Transcriptase with nuclease-Free Water to a final volume of 15 μl. Add 15 µl of revese transcription reaction mix to each reaction tube containing the 5 µl RNA and primer mix. Final volume is 20 µl. Use PCR to finish the RT reaction at 25°C for 5 min, 42°C for 60 min, 70°C for 15 min. The RT-PCR amplification was performed using Maxima™ SYBR Green qPCR Master Mix (2X) kit  with epsilon germline primers or GAPDH primers. Primers were synthesized by Sigma-Aldrich Corporation , see Quantitative real time PCR was used to detect the expression of Xbp-1 and (IgE-H). U266 (1.5 × 106 cells/ml were cultured with formononetin at a concentration of 10 (µg/ml) for 3 days. After the culture time point the cells were harvested, centrifuged at 3,000 rpm for 5 min and the supernatant discarded. The cell suspension was washed with PBS and centrifuged at 3,000 rpm for 5 min. Then the cell lysis was done for protein extraction using 100–200 µl of RIPA lysis buffer, with intermittent vigorous vortexing and incubating on ice for about 1 h. After completion of lysis, the tubes were spined in centrifuge at 14,000 rpm for 20 min at 4 degrees Celsius. The supernatant was collected and transferred to another tube to measure the protein concentration. Electrophoresis was done at 90 V for 15 min followed by 100–110 V for the remaining time and transferred to a PVDF membrane. The membrane was incubated with primary antibodies of the XBP-1, β-actin overnight. The next day, the membrane was washed with PBST and incubated with secondary antibody for 2 h and after the membrane was washed with PBST 4 times before adding the chemiluminescence substrate and imaging. The primary antibodies of XBP-1 and β-actin were ordered from Cell Signaling  and were used at a 1:1,000 dilution, the secondary antibody was used at a 1:10,000 dilution.We used western blotting analysis to measure the protein U266 cells at 3.0 × 10p-values were determined by a two-sided calculation. p value ≤ 0.05 was considered as statistically significant.All statistical analyses were performed using Sigma Stat 3.5  or GraphPad Prism . One way ANOVA  was performed followed by Bonferroni correction for all pairwise comparisons. For skewed data, the differences between the groups were performed by one way ANOVA on rank followed by Donne's method for all pairwise comparisons. p < 0.05; p < 0.001) with an IC50 value of 49.76 µg/ml  were cultured with U266 cells at a concentration of 250 µg/ml each. All three herbs significantly inhibited the production of IgE in U266 cells. Of the three herbs Sophora flavescens had the highest inhibitory effect on IgE production when compared to the others Glycyrrhiza uralensis and Ganoderma lucidum. The IgE inhibition percentage of Sophora flavescens is 96.54% ± 3.01%, while Glycyrrhiza uralensis and Ganoderma lucidum were 33.90% ± 5.63% and 33.35% ± 8.20% respectively (p < 0.001). Further experiment showed that Sophora flavescens dose-dependently inhibited IgE production (p < 0.01) with an IC50 values of 43.13 µg/ml  for 6 days. After the culture time point, the cells were harvested, supernatant was collected and using ELISA IgE concentration was measured. Results showed that ASHMI formula significantly inhibited IgE production in a dose-dependent manner  with an IC50 value of 1.612 µg/ml . We next focused on SF-DB for further study because this fraction contains two major peaks, in addition to its potency of IgE suppression. SF-DC fraction showed one major peak and several small peaks and had very low yield therefore it would be difficult to isolate the pure compound from SF-DC. SF-DD was the last collection of the fraction process  that contains many small peaks; therefore, it would be difficult to isolate pure compounds. Prior to further isolation and purification of SF-DB compounds, we tested SF-DB dose dependent effect on IgE production. It was shown that SF-DB dose-dependently inhibited the IgE production in a non-toxicity manner . Formononetin significantly inhibited the IgE production in vitro at the concentration as low as 0.15 µg/ml. No significant toxicity was observed at concentrations lower than 1.25 µg/ml . No significant cytotoxicity was observed as indicated by trypan blue measure . The results showed that formononetin dose dependently inhibited the IgE production by U266 cells (25 µg/ml . The IC516 µg/ml . Having  measure  and cyto measure . To inve measure .p < 0.001) when compared to the untreated culture group.IgE heavy chain transcription is a critical step for IgE isotype switching. To investigate the mechanism by which formononetin inhibits IgE production, mRNA expression of IgE heavy chain and XBP-1 was measured using real-time PCR. Human U266 cells were cultured with formononetin at 10 µg/ml, the total RNA in U266 cells was extracted and real time-PCR experiment was performed. Results showed that both the relative mRNA expression of IgE Heavy chain and XBP-1 were significantly downregulated by formononetin (p < 0.01).The gene expression of XBP-1 was significantly decreased by formononetin, therefore we used western blotting to measure the effect of formononetin on the protein expression of XBP-1. Our results showed that formononetin significantly inhibited the protein expression of XBP-1  regions. The human ɛ-germline gene promoter region consists of several promoters which includes STAT6 promoter region and NF-ĸB region. NF-ĸB binds to two sites in the ɛ-germline gene promoter and cooperates with STAT6 in synergistic action of transcription (39). The activity of STAT6 is highly influenced by NF-ĸB , 34. At Upon activation mature B cells undergo immunoglobulin class switch recombination and differentiate into antibody-secreting plasma cells . AntagonActivation of ɛ germline gene is an essential step towards IgE expression , 36, whiSophora flavescens, one of the three herbal constituents of ASHMI, is the major component which inhibited the IgE production. Formononetin, isolated from Sophora flavescens, significantly inhibited IgE production in vitro without altering cell viability. The IgE inhibitory mechanisms of formononetin might be through the regulation of XBP-1.We, for the first time, demonstrated that


We utilize a systems biology approach to show the PKC life cycle is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types: maturation via processing of newly synthesized enzyme and secondary messenger-dependent activation of dormant, but catalytically competent enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and stored in the cytosol as a stable, signaling-competent inactive and autoinhibited molecule. Upon extracellular stimulation, diacylglycerol (DAG) and calcium ion (Ca2+) generated at the membrane bind PKC. PKC then undergoes cytosol-to-membrane translocation and subsequent activation. Our model shows that, once activated, PKC is prone to dephosphorylation and subsequent degradation. This model also describes the role of HSP70 in stabilization and re-phosphorylation of dephosphorylated PKC, replenishing the PKC pool. Our model shows how the PKC pool responds to different intensities of extracellular stimuli? We show that blocking PHLPP dephosphorylation replenishes the PKC pool in a dose-dependent manner. This model provides a comprehensive understanding of PKC life cycle regulation.Protein kinase C (PKC) enzymes are a family of kinases that mediate signal transduction originating at the cell surface. Most cell membranes can contain functional PKC enzymes. Aberrations in the PKC life cycle may result in cellular damage and dysfunction. For example, some cancerous cells exhibit alterations in PKC activity. Here, we use a systems biology approach to describe a molecular model of the PKC life cycle. Understanding the PKC life cycle is necessary to identify new drug targets. The PKC life cycle is composed of three key regulatory processes: maturation, activation, and termination. These processes precisely control PKC enzyme levels. This model describes the fate of PKC during  Cell membranes are platforms for the transduction of vital signals affecting cellular fate . Signal 2+/DAG transmit crucial signaling information that controls cellular fate. Members of this kinase family bind to secondary messengers, which may play a critical role in translating environmental cues into useful and vital intracellular information by determining the phosphorylation status of key downstream substrates. Intriguingly, the phosphorylation events within the members of this family are constitutive whereas the dephosphorylation processes are agonist-driven  phosphorylation controls PKC enzyme stability; and 2) PKC activation and termination are agonist-modulated events . AccordiThe protein conformation of these family members is under tight control. Conformational control is necessary for PKC to function in a multitude of cell types. Two key molecular events may modulate PKC conformation: 1) events of constitutive phosphorylation; and 2) secondary messenger-dependent membrane binding/unbinding events. Newly synthesized PKC is unstable and quickly undergoes degradation when not modified by phosphorylation events. Naïve PKC undergoes a series of ordered phosphorylation events to avoid degradation events . These p2 for phosphorylation to occur at the hydrophobic motifs of PKC  Depleted PKC has been observed in cells lacking kinases, such as PDK1 . 2) In as of PKC . 4) It is of PKC . 5) Susts of PKC . 6) Inhis of PKC . 7) PH ds of PKC . 8) In ts of PKC . 9) Finas of PKC .Although the basic life cycle model described here reflects one pathway of PKC life cycle, studies indicate that basic model can be extended by incorporating additional complexities. In certain PKC signaling cases the fully phosphorylated form of PKC can also be directly targeted for degradation . In thatde-novo synthesis. In addition, we explore possible molecular mechanisms responsible for PKC activation and down-regulation during agonist-induced stimulation. Understanding the PKC life cycle is crucial to designing therapeutics targeting PKC enzymes to alleviate many human diseases. PKC dysfunction has been correlated with a multitude of disease states. For example, PKCβ protein levels are 4-5-fold higher in breast cancer cells than in normal mammary cells , the complex of PKC. PA with mTORC2 at the membrane (C.PKC.PA), the enzyme-complex phosphorylated at activation, hydrophobic and turn motifs and located in the cytosol (C.PKC.PA.PH.PT); the activated and phosphorylated enzyme at the membrane (PKC.PA.PH.PT)A; and the dephosphorylated but active molecule located in the cytosol (PKCA). Here, C.PKC.PA.PH.PT is cytosolic and on activation stimulation translocates to membrane. The active form of enzyme at membrane PKC.PA.PH.PTA undergoes PHLPP mediated dephosphorylation and active but dephosphorylated form at membrane i.e., PKCA translocate back to cytosol where it can undergo degradation or re-phosphorylated to become part of a stable pool of the enzyme. According to this model, brief protein synthesis stimulation leads to generation of naïve PKC. Newly synthesized, unphosphorylated PKC is highly unstable and only loosely tethered at the cell membrane. Naive PKC is an exposed pseudo-substrate with an accessible C-terminal tail. According to the model, constitutively active PDK1 docks with the C-terminal of nascent PKC and modulates PKC phosphorylation at activation loop (PA). Constitutive phosphorylation stabilizes the previously labile naïve enzyme and triggers binding to the mTORC2 complex at the cellular membrane. This binding event, in turn, catalyzes a series of sequential autophosphorylations on PKC’s C-terminal tail, hydrophobic motif and turn motif. These phosphorylation events are PH and PT. These three constitutive ordered phosphorylation events generate a mature, phosphatase/protease resistant, but catalytically competent molecule denoted as C.PKC.PA.PH.PT. Subsequently, the directions are provided to catalytically competent molecule for cytosolic localization and extended storage. The C.PKC.PA.PH.PT form of PKC is stable and inactive, but catalytically competent and functionally responsive to second messenger changes inside the cell or at the membrane compartment. According to this model, the first phase of the PKC life cycle involves only constitutive phosphorylation events. During this phase, after a brief protein synthesis pulse, the naïve molecule undergoes transformation into a stable and inactive specie but is still responsive to elevation in the levels of second messenger at the specific membrane locations. In the first phase of the life cycle, PKC is stored at many discrete locations throughout the cytosol. Our model emphasizes how PKC functionality and signaling depends on constitutive processing events, leading to its stabilization and catalytic competence?In this study, we propose a model for the PKC enzyme life cycle. This model explains the fate of the nascent PKC molecule. We propose  that tem2+ and DAG or phorbol myristate acetate (PMA). A brief stimulation pulse at the membrane either generates second messengers Ca2+/DAG or PMA. The model is based on prevailing evidence that enzyme reversibly activated by Ca2+/DAG binding but irreversibly activated by PMA binding. Thus, the model states that usually Ca2+/DAG binding is not strong enough to promote degradation however, PMA binding can lead to significant degradation and down-regulation. Thus, this model states that low intensity pulse stimulation represents Ca2+/DAG binding which leads to reversible activation and on the removal of Ca2+/DAG, the enzyme is released from membrane and adopts the stable, autoinhibited conformation in the cytosol whereas, the high intensity pulse stimulation represents phorbol esters binding leading to irreversible activation and degradation. Thus, the lower intensity activation pulse, in turn, leads to translocation, reversible activation, and re-translocation to cytosol while higher intensity activation pulse leads to translocation, irreversible activation, dephosphorylation and either, degradation in cytosol or restoration back to dormant form. The model shows that a brief pulse of DAG or PMA in the membrane compartment leads to the translocation of mature PKC from the cytosol to the membrane. At the membrane, binding with DAG/PMA converts inactive PKC to active, but phosphatase-sensitive PKC. This form of PKC is represented by PKC.PA.PH.PTA. In this stage, PKC is active but prone to dephosphorylation. PHLPP-mediated dephosphorylation then relocates PKC back to the cytosol. Active but dephosphorylated PKCA is also a target either for degradation or re-phosphorylation by HSP70-mediated rescue and autophosphorylations. Our model suggests that chronic activation of PKC may result in complete dephosphorylation and down-regulation of PKC. PKC’s life cycle involves de-novo synthesis, maturation, activation and inactivation/re-migration or termination. Each of these events affects PKC levels and, thus, the agonist-mediated amplitude and duration of active PKC signaling.This model suggests that, during the second phase of the life cycle, inactive, phosphatase/proteasome-resistant PKC species undergo activation either through second messengers CaMaterials & Methods: R1 -R12) describing the basic life cycle model are obtained through a systematic strategy described here: 1) following rate constants were obtained from literature: 1) half-time of PDK1-mediated phosphorylation event of nascent, endogenous PKCα enzyme in COS-7 cells at activation loop  degrion loop ; 3) degrion loop ; 4 half-ion loop ; 6) the ion loop ; 7) The ion loop ; 8) halfion loop ; 2) The ion loop . One mayion loop 1–S18 ofde-novo PKC synthesis, and the system is fixed in the basal state  and is linked to red dashed line representing (1); intensity set at 0.005 (nM) and is linked to red dashed line (2); intensity set at 0.05 (nM) and is linked to red dashed line (3) and activator (PMA) intensity set at 0.5 (nM) and is linked to red dashed line (4). These different strengths of second messenger/PMA pulse indicate different levels of PKC activation  and varied the duration of this low-level pulse. In these simulations  we used 17. Our results show that blocking dephosphorylation prevents down-regulation of cPKC even at higher stimulation levels  during simulations . The strength of these sequential pulses was 0.5 (nM) with a fixed duration of 15 min. During these activation pulses dephosphorylation is inhibited by completely blocking the parameter kA.PH.PTA and PKCA . With no PHLPP blocking cPKC is completely down-regulated . Some of the rate constants in this biochemical reaction system are obtained through limited available experimental data , of PKC is matched closely to experimentally observed data points  can be perturbed either through stimulation of protein synthesis or through secondary-messenger Ca2+/DAG-modulated reversible or PMA-modulate irreversible activation. New PKC synthesis is described by equations R1 and R2. , and undergoes maximum phosphorylation at its activation site  as a result (4). The phosphorylated enzyme (PKC.PA) forms a biochemical complex (C.PKC.PA) with the mTORC2 complex (5). Phosphorylation at activation site PA reduces the degradation rate of naïve enzyme because PKC.PA is more stable and degrades more slowly as described by R6. In contrast, λ2 represents the degradation rate of this enzyme species . The active C.PKC.PA.PH.PTA form of the enzyme is prone to rapid dephosphorylation by phosphatases PHLPP1 and PHLPP2. These dephosphorylation events are described by equation R10 . Heat shock proteins HSP-60 and HSP-70 act on the active and dephosphorylated form of the enzyme, rescuing and recovering PKC from degradation pathways and restoring the pool of inactive, catalytically competent PKC in the cytosol (12).The PKC life cycle model described by the biochemical reaction equations , R1-R12  and R2. . These rd R2. (1 . The loa protein . During anscript . This trcript (2 . The naïpt (2 (3 . However species . The C.P R7 & R8 . The ination R10 . Upon deDuring simulations, the induction of new proteins was simulated by a 10-min pulse of protein synthesis, which produced an increase in the total PKC protein concentration.Biochemical Reactions, is used to generate differential equations through accounting the mass balance of each biochemical specie as described in above equations R1-R12. The differential equations provide the temporal variations in the concentration of each specie participating in above reaction network i.e., R1-R12. The differential equations (1-R12 could be stiff as some solutions may vary slowly but some other solutions might vary rapidly. Some of the dynamical coefficients’ values were estimated from experimental data such as degradation rates, and rates of constitutive and autophosphorylations. Unknown rate constants were scaled to obtain dynamics that were comparable to experimental values (The law of mass action as described in discussion section and above in quations  resultinl values . Unless


Portal vein system thrombosis (PVST) is a common postoperative complication brought by laparoscopic splenectomy and pericardial disconnection (LSD) among patients who suffered from portal hypertension and hypersplenism. This research lies mainly in probing into the risk factors of PVST and evaluating the effects of warfarin on PVST prevention.n = 68) and aspirin group (n = 63). Meanwhile, thrombosis factors were analyzed in PVST arm (n = 48) and non-PVST arm (n = 83).We took 131 individuals who have carried out LSD from January 2015 to January 2021. Patients were divided into warfarin group . Risk factors for PVST were analyzed, and diabetes, the diameter of the portal vein and splenic vein, and the velocity of portal blood flow were statistically significant between the PVST arm and non-PVST arm (P <  < 0.05). Multiple logistic regression analyses have shown that diabetes, portal vein diameter, splenic vein diameter, and the velocity of portal blood flow were the risk factors of PVST.We analyzed the early postoperative anticoagulation effect, 20 patients (29.4%) in the warfarin group developed PVST, and 28 patients (44.4%) in the aspirin group. The chance to PVST during the first year after operation was lower in the warfarin group than in the aspirin group (The portal vein diameter, splenic vein diameter, portal vein flow velocity, and diabetes are risk factors for the PVST after LSD. The prophylactic use of warfarin anticoagulation markedly decreases the probability of occurrence of the PVST in patients with portal hypertension after LSD compared to aspirin.The online version contains supplementary material available at 10.1007/s00464-022-09340-5. Liver cirrhosis is a frequently occurring chronic liver disease, which is usually asymptomatic in early stage and portal hypertension in later stage . In patiPVST means a thrombosis occurring in the splenic vein, portal vein, superior mesenteric vena, or intrahepatic portal vein, the most common of which is the thrombosis occurring in the splenic vein . PVST caDespite the fact that many studies have shown that routine prophylactic anticoagulant therapy after splenectomy can reduce the risk of PVST formation, it is unclear which anticoagulants are effective in preventing PVST after LSD. It has been suggested that common antiplatelet drugs like aspirin and clopidogrel are ineffective in preventing PVST . HoweverHence, the current research targeted to argue the major factors for PVST and evaluate the effectiveness of warfarin in preventing PVST formation following LSD. From January 2015 to January 2021, we compared the effectiveness of warfarin and aspirin in the postoperative prophylaxis of PVST in patients with portal hypertension who underwent LSD surgery at our center.All individuals with cirrhosis hypersplenism admitted to the Department of Hepatobiliary Surgery, Affiliated Hospital of Guizhou Medical University, and The Affiliated Cancer Hospital of Guizhou Medical University from January 2015 to January 2021 were recruited. The admission criteria were as follows: The research object was aged from 18 to 80 years and had been diagnosed with portal hypertension and hypersplenism; History of esophagogastric variceal bleeding and Childe-Pugh A or B liver function did not have PVST, and detected by CT, MRI, or Doppler ultrasound before operation; and successfully underwent LSD.The exclusion criteria were as follows: The patient was diagnosed with liver cancer or any other malignant tumor; there are coagulopathy or hypercoagulable unrelated to liver disease; there is evidence of clot within the portal vein system prior to undergoing the LSD procedure; combined with serious angiocardiopathy, lung, kidney dysfunction or blood system disease; baseline international normalized ratio (INR) > 2; pregnancy; previous history of the cardiovascular and cerebrovascular hemorrhagic disease: infected with immunodeficiency virus.In this study, patients in both groups were subcutaneously injected with low-molecular weight heparin  (4250 U/d) for 5 days and orally dipyridamole  25 mg 3 times a day for 3 months from the POD1. From postoperative day (POD) 3, the experimental group was given warfarin sodium  orally for 6 months. The pre-dose was 2.5 mg/d, and the dose was quickly adjusted to achieve the target INR of 2–3. The control group was given enteric-coated aspirin tablets  orally at 100 mg/d on the POD3 for 6 months. Warfarin or aspirin should not be used if PVST does not occur within 6 months after surgery. All the patients have completed clinical observation during the 18 months of follow-up.Once PVST was confirmed after LSD, the patients would receive a thrombolytic therapy. Urokinase was administered via the peripheral vein with a bonus dose of 250,000 units within 10 min, followed by a continuous infusion of 5000 units/h for 48 ~ 72 h via a micro-infusion pump. Following the thrombolytic treatment, the patients were administrated with oral warfarin or aspirin plus dipyridamole according to the different groups. The drug doses were adjusted based on the INR and PLT levels.Preoperative information was retrospectively collected, including gender, age, body mass index (BMI), diabetes, Childe-Pugh classification, leukocyte (WBC) count, platelet count (PLT), total bilirubin (TBIL), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (CRE), INR, fibrinogen (FIB), spleen longitudinal diameter, portal vein diameter, splenic vein diameter, and speed of portal blood flow. Intraoperative messages included duration of operation and estimated intraoperative blood loss. Postoperative materials included WBC, PLT, TBIL, ALT, BUN, CRE, INR, FIB, and postoperative anticoagulant treatment regimen.Participants were randomly assigned to warfarin group and aspirin group (warfarin group odd and aspirin group even) according to the order in which they were enrolled. Patients were treated with warfarin or aspirin plus dipyridamole according to the different groups. Ultrasound and radiologists were blinded to the randomization results. The present study had approval from the Human Research Ethics Committee at the Affiliated Hospital of Guizhou Medical University. All sufferers were informed and consented.The patients were placed in supine position. The operation was performed under general anesthesia. A laparoscopic trocar was inserted into the umbilicus through a 12 mm incision, and the pneumoperitoneum was set to 9 ~ 10 mmHg. Following the successful establishment of the pneumoperitoneum, three additional trocars were inserted at the level of the subxiphoid, the left subcostal margin midclavicular line, and the left flank level. First, pericardial devascularization was using a LigaSure vessel-sealing instrument . Then splenogastric ligaments, splenocolic ligament, and lienorenal ligament were divided by LigaSure. In the tail of the pancreas, the splenopancreatic fascia was cut and identified. With an endoscopic linear stapler, splenic hilar was performed. The spleen was removed from the umbilical incision, placed in a plastic bag, and removed.PVST formation refers to venous thromboembolic events of the splenic vein, the main branch of the portal vein, and the intrahepatic branch vein. The primary endpoint of this work was PVST. The patients routinely underwent enhanced CT 3 days before the operation. Ultrasound examinations were performed at 1, 2, 4, and 12 weeks after surgery, as well as every three months after that. However, due to the influence of the operator's tech and the patient's body habitus, thrombus may not be detected by ultrasound scanning. Therefore, a contrast-enhanced CT scan is to confirm the diagnosis if the ultrasound reveals suspicious echogenic material or reduced or no blood flow in the portal venous system lumen . The locP value < 0.05 was defined as having statistical significance. The statistical package for the Social Sciences (SPSS) Version 16 software  works for statistical analysis.Files are depicted as mean (standard deviation), median (interquartile range), or percentage. Dual-tailed non-paired Student’s t-test was applied for structural comparison. After the chi-square test, the classified data were expressed as frequencies. The factors of PVST occurrence were discussed by single-variable analysis, and then meaningless variables were gradually removed while the logistic regression model for used in multivariate analysis. A n = 69) or aspirin group (n = 67). One patient stopped warfarin on POD5 due to bloody stools; four patients stopped aspirin treatment on POD7 due to gastrointestinal reactions. Finally, 131 patients completed treatment, including the warfarin group (n = 68) and aspirin group (n = 63).From January 2015 to January 2021, all 156 individuals with portal hypertension complicated and secondary hypersplenism received LSD in our center. Twenty patients were excluded based on enrollment conditions in this study. The other 136 patients were randomly allocated to the warfarin group , WBC, PLT, TBIL, ALT, BUN, CRE, INR, FIB, spleen longitudinal diameter, portal vein diameter, splenic vein diameter, speed of portal blood flow, duration of surgery, and intraoperative bleeding  in the first year after operations were substantially lower in the warfarin team (P > 0.05).Then we analyzed the clinical baseline traits of the PVST and non-PVST arms in Table P < 0.05; Table P < 0.05), a broader splenic vein diameter (P < 0.05), and a slower speed of portal blood flowing (P < 0.001). Files have been performed by logistic multivariable regression, and certain statistically significant variables covered portal vein diameter, splenic vein diameter, and speed of portal blood flowing , suggesting that platelets cannot be a separate pathogenic factor for PVST formation when LSD has been done. This result may be related to prophylactic anticoagulant therapy after the operation.In patients with liver cirrhosis complicated with portal hypertension, the total number of platelets decreases, mainly due to the increase of platelet reserve and slowing down of platelet velocity during splenomegaly, allowing enough time for them to contact splenic macrophages . As a reThe diameter of the portal vein has been mentioned several times as a potentially dangerous factor in PVST formation . This isAt present, prophylactic anticoagulant therapy for PVSD after LSD has been in a dilemma for a long time, because the portal vein system may still have thrombosis in the presence of decreased platelet count and increased INR. The most concern was that early postoperative use of anticoagulants in patients with cirrhosis may increase the risk of postoperative bleeding due to the inner blood coagulation disorder. Therefore, the prophylactic anticoagulant therapy for PVST after LSD is still controversial, mainly based on personal experience. However, recent studies have proved that prophylactic anticoagulant therapy is safe and feasible, which can effectively reduce the occurrence of PVST and reduce the risk of bleeding , 27. TheBased on our 1-year randomized controlled study involving patients with cirrhosis after LSD, prophylactic use of warfarin has been proven to be effective and safe. Warfarin treatment can significantly reduce the happenchance of PVST following surgery, in contrast with aspirin treatment. In this study, we realized that the chance of PVST in warfarin teams occurred fewer than the aspirin ones at all-time points from POD7 to POM12, and the difference between the two groups from POM1 to POM6 was statistically significant. Finally, compared to aspirin, earlier postoperative use of warfarin significantly reduced the occurrence rate of PVST.The work implemented as a single-center-based cohort study enlisted a comparatively small sample capacity, so the consequences had potential limitations. Our study subjects were cirrhotic patients with portal hypertension in Guizhou, China, and the results may have limited external validity in different regions and Settings. In addition, it is difficult for some patients to achieve the target value of 2–3 during oral warfarin adjustment of INR, which brings potential difficulties for clinical application.In conclusion, PVST is familiar postoperative comorbidity of LSD; furthermore, its incidence might be reduced by early postoperative anticoagulation. The anticoagulation effect of warfarin is better than that of aspirin. The portal vein diameter, splenic vein diameter, and portal vein flow velocity are predictors of PVST occurrence. Close postoperative follow-up and early anticoagulation are critical for patients who are at high risk of venous thrombosis formation.Supplementary file1 (DOCX 18 kb)Supplementary file2 (DOCX 18 kb)Below is the link to the electronic supplementary material.