Background: The three-dimensional (3D) geometry of coronary atherosclerotic plaques is associated with plaque growth and the occurrence of coronary artery disease. However, there is a lack of studies on the 3D geometric properties of coronary plaques. We aim to investigate if coronary plaques of different sizes are consistent in geometric properties.Methods: Nineteen cases with symptomatic stenosis caused by atherosclerotic plaques in the left coronary artery were included. Based on attenuation values on computed tomography angiography images, coronary atherosclerotic plaques and calcifications were identified, 3D reconstructed, and manually revised. Multidimensional geometric parameters were measured on the 3D models of plaques and calcifications. Linear and non-linear  fittings were used to investigate the relationship between multidimensional geometric parameters . Pearson correlation coefficient (r), R-squared, and p-values were used to evaluate the significance of the relationship. The analysis was performed based on cases and plaques, respectively. Significant linear relationship was defined as R-squared > 0.25 and p < 0.05.Results: In total, 49 atherosclerotic plaques and 56 calcifications were extracted. In the case-based analysis, significant linear relationships were found between number of plaques and number of calcifications  as well as total volume of plaques , between number of calcifications and total volume of plaques  as well as total volume of calcification , and between the total volumes of plaques and calcifications . In plaque-based analysis, the power function showed higher R-squared values than the linear function in fitting the relationships of multidimensional geometric parameters. Two presumptions of plaque geometry in different growth stages were proposed with simplified geometric models developed. In the proposed models, the exponents in the power functions of geometric parameters were in accordance with the fitted values.Conclusion: In patients with coronary artery disease, coronary plaques and calcifications are positively related in number and volume. Different coronary plaques are consistent in the relationship between geometry parameters in different dimensions. Coronary artery disease is a cardiovascular disease that has been the leading cause of death globally  scan is the most commonly used medical imaging technique in the diagnosis of CAD , size, diameter, and composition of coronary artery plaques . For example, sphericity has been proposed to evaluate the geometric consistency between different objects. It was defined as the ratio of the surface area of a sphere, which has the same volume as the given particle to the surface area of that particle  in left coronary artery. In total, 19 patients  were finally included for analysis, as listed in The 3D geometry of coronary arteries and plaques was reconstructed from the CCTA images using the software MIMICS 20.0 . The reconstruction method was semiautomatic, using the Coronary Segmentation Tool of Mimics Medical Suite. Firstly, the position of the aorta was marked. Then, with start and end points marked on the CCTA image dataset, an arterial segment can be automatically reconstructed with the result saved in an independent set (a “mask” in MIMICS). For the left coronary artery tree, artery segments were extracted from the left main coronary artery (LM) to major branches, including the left anterior descending artery (LAD) and left circumflex artery (LCX), and, finally, the distal branches. The small branches  were trimmed off. The left coronary artery tree was derived by connecting artery segments using the union operation of different sets.The 3D reconstruction of coronary plaques was based on a semiautomatic two-step approach. It is difficult to distinguish between fibrotic, fibro-fatty, and fatty atherosclerotic lesions. Therefore, in this study, the plaque components were categorised as calcified  and non-calcified. Firstly, based on the position of plaque on the two-dimensional (2D) CCTA images, the spatial ranges of plaque were set in three directions . In the 3D space defined by the ranges, the plaque was automatically extracted using thresholds of CT attenuation value . Due to the difference in plaque composition, the CT attenuation value varies among different scans. Therefore, the thresholds were set patient specifically. Low attenuation values (0–150 HU) were used to identify non-calcified plaques, whereas high attenuation values  were used for calcified components. Firstly, the calcifications were automatically extracted . The demIn the second step, the plaque geometry was further modified. Considering the blooming artefact caused by calcification, the surrounding non-calcified components have higher HU values. The boundaries between non-calcified components and calcification or artery lumen were checked and adjusted manually. The areas opposite to the calcification were also reviewed to add the non-calcified plaque components that were not detected by the threshold used in the first step due to the effect of surrounding tissues  on the whole plaques and calcifications, respectively.For each plaque and calcification, firstly, the volume and the surface area were measured automatically. Secondly, the length, surface distance, cross-sectional area, and diameter were measured manually. The length of a plaque or calcification was defined as the longest straight-line distance between its proximal and distal ends. The surface distance was defined as the length of the shortest path on the surface between the proximal and distal ends. For the whole plaque, the cross-section area was measured on the cross-section perpendicular to the local centerline of the arterial segment with the maximal area . The croThe statistical analysis was performed using SPSS 24.0 software  based on each case, plaque, and calcification, respectively.r), coefficient of determination (R-squared), and p-value calculated. A significant linear relationship was defined as r > 0.5 (R-squared > 0.25) and p < 0.05. A strong linear relationship was defined as r > 0.8 (R-squared > 0.64) and p < 0.05.In the case-based analysis, the sum of volumes of different plaques or calcifications was calculated for each subject. The relationships among the numbers and volumes of plaques and calcifications were investigated using simple linear regression with Pearson correlation coefficient  of plaques. Similarly, in the calcification-based analysis, simple linear regression analysis was performed on the geometric parameters measured from each calcification . If any significant linear relationship was found between two parameters of different dimensions , the non-linear regression analysis based on power function was performed. The results of linear and non-linear regressions were compared using their R-squared values. Finally, to further investigate the relationship between plaques and calcifications in volume, simple linear regression analysis was performed in the plaque-based analysis between the plaque volume and the total volume of calcifications in the plaque.Figure 6). To evaluate if the models can accurately reflect the plaque geometry, the ratio between plaque length, surface area, and volume was calculated and compared with the obtained measurement results.Based on the analysis of multidimensional parameters (see Results), we proposed two models to describe the geometry of small (patchy) and big (circular) coronary plaques. All the plaques were simplified as a part of a cylindrical shell with uniform thickness , LCX (in 7 subjects), LM (in 5 subjects), as well as the diagonal (in 4 subjects), and marginal (in 3 subjects) branches. The CA scores ranged from 1.2 to 856.4. Therefore, the plaques in this study were diverse in location and composition.In the 19 subjects, in total, 49 atherosclerotic plaques and 56 calcifications were extracted from the left coronary arteries. In each case, the numbers of plaques and calcifications were (mean ± SD) 2.579 ± 1.387 and 3.158 ± 1.803; the mean calcification volume and mean whole plaque volume were 24.137 ± 24.156 r = 0.650, p = 0.003), between the number of plaques and the volume of plaques , between the number of calcifications and the volume of plaques , and between the number of calcifications and the volume of calcifications . The volume of plaques and volume of calcifications showed a strong linear relationship   .r = 0.506, p < 0.001). Strong linear relationships were found between the volume and the surface area , volume and length , volume and surface distance , surface area and length , surface area and surface distance , length and surface distance , as well as cross-section diameter and cross-section area . There was a significant linear relationship between the plaque volume and the total volume of calcifications in each plaque .For the plaques, a significant linear relationship was found between the volume and the cross-section area (R-squared: 0.256 and 0.236 for linear and non-linear regressions), as well as between the length and the surface distance where the values are very approximate (R-squared: 0.993 and 0.990 for linear and nonlinear regressions).In r = 0.895, p < 0.001), volume and surface distance , as well as length and surface distance . In R-squared value than the non-linear regression. All the relationships in In the calcification-based analysis, strong linear relationships were found between volume and length  locates along the external wallHypothesis 1.4: the plaque length (L) goes across the geometryIn both cases, it can be derived that In the early stage of plaque formation, the vessel wall thickening often emerges from one side of coronary artery and extends both longitudinally and circumferentially. Therefore, we proposed a model that describes the geometry of an early-stage plaque that covers circumstantially a part of the artery wall  of coronary plaques is a novel finding. As we observed, coronary plaques often grow along the artery wall, forming curved shells, rings, or more complicated shapes, which are quite different from a sphere . TherefoOn the other hand, geometrical parameters of coronary plaques can provide information about many CAD-related clinical risks: the severity of ischemia, the risky areas of plaque growth, and the stability of arteriosclerotic plaques , the Zhejiang Provincial Department of Science and Technology (Grant No. 2020C03016), and the Basic Research Foundation of Shenzhen (Grant No. GJHZ20180928155002157).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Factors that influence utilisation rates of patient reported outcome measures (PROMs) for low back pain (LBP) within the chiropractic profession of Australia are currently unknown. This study aimed to examine whether factors, including age, sex, experience level, clinical title , or a clinicians’ perceived value of PROMs, are predictive of the frequency and/or type of PROMs used by chiropractors in the management of LBP.A cross sectional online survey was distributed to members of the Chiropractic Association of Australia  and Chiropractic Australia (CA). 3,014 CAA members and 930 CA members were invited to participate totaling 3,944, only respondents that were using PROMs were included in the analysis (n = 370). Ordinal logistic regression was used to examine associations between clinician demographics and perceived value of PROMs, and the frequency of pain, health, and functional patient reported outcome measure (PROM) usage by chiropractors.Principal chiropractors were more likely ) than associate chiropractors to frequently use pain-related PROMs for the management of patients with LBP. The remaining demographic factors  were not associated with the frequency of PROM usage; nor were the perceived value clinicians place on PROMs in clinical practice.Principal chiropractors were more likely to frequently use pain-related PROMs for the management of patients with LBP when compared to associate chiropractors. Demographic factors, appear to have little influence on PROM usage. While chiropractors place high value on PROMs, these beliefs are not associated with increased frequency of PROM usage for the management of LBP. Patient reported outcome measures (PROMs) are tools used to measure a health status or condition from the perspective of the patient  and inclPROMs are recommended in clinical guidelines for a range of health disciplines and patient populations but have been most widely developed and adopted in the physiotherapy management of LBP , 12, 13.A recent study of Dutch physiotherapists examined what factors influence the utilisation of PROMs in physiotherapy private practice . The stuResearch on PROM utilisation within the chiropractic profession is emerging. A survey of chiropractors in Canada reported that 85% and 89% routinely used a health-related and pain-related PROM, respectively, at every initial consultation . SimilarThis study used survey data collected as part of a previously published study . A detaiThe original survey included 25 items that aimed to establish the type and frequency of PROM usage in LBP, and any potential barriers and facilitators to their use. For the current study, only surveys from respondents that answered, ‘Yes’, to the question “Do you use PROMs in practice?” were included in the analysis (n = 370). From these surveys the following demographic variables were obtained: 1) sex ; 2) years in practice; and 3) clinical title . The clinical title ‘associate’ refers to an employee or contractor who works within a private practice, whereas ‘principal’ chiropractor refers to the senior practitioner, usually the clinic or practice owner.In addition, the perceived value that PROMs had on respondents’ clinical practice were obtained using three questions scored on a 5-point Likert scale Table . TherefoThe type and frequency of PROMs used was obtained by asking the question “Which < PROM name > for LBP do you use, and how frequently?”. In the original survey this question had 8 response options: each visit, initial, weekly, every 3–6 visits, monthly, every 6–9 visits, every 9–12 visits, annually or never. For the current study, these responses were recoded into a 5-point Likert scale to describe the frequency of PROM usage (Table 2) was included to assess the statistical significance of the explanatory variables in the statistical model, testing the null hypothesis that the model coefficients are equal to zero. All data were analysed using SPSS software version 22, with statistical significance set at p ≤ 0.05.Participant characteristics and question frequency response data are expressed using descriptive statistics. Univariate ordered logistic regressions with proportional odds were used to determine which of the seven independent variables  were associated with the frequency of PROM use  for each category . Multivariate ordinal logistic regressions were planned when two or more variables with p < 0.2 were found from the univariate analyses. Regression coefficients were expressed as odds ratios (OR) with 95% confidence intervals (95% CI) to indicate the strength of the explanatory variable to the dependent variable. The Wald Chi-Square statistic  of 15.9 years (11.2). The frequency distribution (%) of age ranges (years) were: 20–29 (16.8%), 30–39 (31.1%), 40–49 (24.9%), 50–59 (16.2%), 60–69 (9.2%), and 70 + (1.9%).Response data for the importance of PROMs to the practitioner and patient, and influence on treatment, are presented in Table A summary of the response data for the frequency of PROM usage for each category is presented in Table 2 = 4.101, p = 0.04, OR = 1.4 (1.0–2.1)) to more frequently use pain-PROMs, compared to associate chiropractors. There was no influence of clinical title on the frequency of health-PROM or functional-PROM use. Similarly, there were no associations between PROM usage and the importance of PROMS to the practitioner or patient, or whether they influence treatment. As only one univariate association had a p-value < 0.2, no multivariate ordinal regressions were performed.The results of the univariate logistic regressions are summarized in Table The aim of this study was to examine whether clinician demographics and/or perceived value of PROMs were associated with the frequency of PROM usage by chiropractors when managing patients with LBP. Although all survey respondent stated they use PROMs for patients with LBP, the data indicate utilisation rates were generally low, with most respondents reporting they either ‘never’ or ‘rarely’ use pain, health, or functional PROMs. Not surprisingly there were no consistent or strong predictors of increased PROM usage for any of the dependent variables. Our broad hypothesis that the value clinicians place on PROMs would influence the frequency of PROM usage was therefore not supported, despite most respondents reporting PROMs were either ‘important’ or ‘very important’ to the practitioner, patient, or when planning treatment. We did, however, find some evidence that clinical title influenced the frequency of PROM usage, with principal chiropractors more likely to use pain-PROMs more frequently than associate chiropractors.Other than clinical title , none of the demographic factors examined had a significant influence on the frequency of PROM utilisation by chiropractors for the management of LBP. Our findings are broadly consistent with research in physiotherapists and suggest that clinician demographics have little influence on PROM usage. Age was not a significant predictor of the frequency of PROM usage. Although the mean odds ratios for age were above 1.0 for each PROM category, none reached the level of significance. Similar findings have been reported in physiotherapy , 18. ForWe assessed whether clinical title  may influence the frequency of PROM usage by chiropractor on the basis that principal chiropractors may have more clinical experience and familiarity with PROMs, and therefore would be more likely to routinely use them in clinical practice. Clinical titles are more commonly used within the chiropractic profession, with ‘associate’ chiropractor referring to an employee or contractor who works within a private practice, whereas ‘principal’ chiropractor refers to the senior practitioner, usually the clinic or practice owner. Hence, there is no literature in other health professions to directly compare our results. We found that principal chiropractors were 1.4 times more likely to more frequently, use pain-PROMs compared to associate chiropractors. This finding cannot be generalised to other types of PROMs as clinical title was not a significant predictor of either functional- or health-PROM usage. While difficult to determine, this may reflect the senior role the principal chiropractors have in guiding clinic policy and procedures, and their role as a mentor and role model for associate chiropractors. Hence, it is possible that principal chiropractors may place higher value on the importance of PROM for the management of LBP, compared to associate chiropractors, and more frequently implement PROMs in clinical practice; perhaps to encourage their use by associate clinicians.It was assumed that a chiropractor’s perceived value of PROMs would influence how frequently they would them, such as higher levels of importance placed on PROMs would be associated with higher rates of PROM usage. In contrast, we found no clear and consistent evidence that PROM utilisation rates are influenced by the value that clinicians place on PROMs for either the clinician, patient, or when planning patient management. This was despite the finding most respondents reporting that PROMs are ‘somewhat important’, or ‘very important’, for the practitioner, patient, and when planning treatment. The disconnect between the perceived value of PROMs and their usage in chiropractic practice appears to contrast research in other allied healthcare professions , whereby a bi-directional relationship between a clinicians perceived values and use of outcome measurement has been found, such that a lack of perceived value was associated with decreased likelihood, and vice versa for greater perceived value , 19–21. While difficult to determine, the relatively low utilisation rates of PROMs in the current study may have prevented strong association being observed using ordinal data , as opposed to the nominal data  used in the physiotherapy studies. Nevertheless, our results suggest that while most chiropractors have a favourable view towards PROMs for the management of LBP, and that is consistent with other allied health professions , very feThe findings from this study have implications on future research in this field, which could include developing education strategies aimed at increasing rates of PROM usage by chiropractors, with an initial emphasis on developing knowledge and improving ease of access. Designing education packages that promote PROM knowledge and behavioral change, rather than beliefs, might be effective at improving the rates of PROM usage in chiropractic practice and may be transferrable to other allied health professions.Furthermore, based on the findings of Copeland et al. 2008 , whereby.This study has several limitations. First, online surveys even when directed towards target populations such as registered health professionals  are suscFuture studies should include a more expansive set of independent variables, based on existing literature in other health professions, to fully understand what factors drive PROM usage by chiropractors for the management of the LBP. Finally, our findings are limited to Australian chiropractors and based on a small selection of LBP PROMs, and so our results cannot be generalised to other countries, body regions/conditions, or PROMs not used in this study.Principal chiropractors are more likely to frequently use pain-related PROMs for the management of patients with LBP when compared to associate chiropractors. Although chiropractors place high value on the importance of PROMs for the management of LBP, these beliefs do not translate to increased frequency of PROM usage. Factors, including age, sex, and experience level, also appear to have little influence on PROM usage.


EC − ED = 1.4 eV) to shallow donor (EC − ED = 0.2 eV) surface traps are thoroughly studied for the donor concentration of 1011 to 1016 cm−2. This study involves an aluminum concentration variation from 5 to 50%. This paper for the first time presents the comprehensive TCAD study of surface donor and analysis of electron concentration in the channel and 2DEG formation at AlGaN–GaN interface.The presence of surface traps is an important phenomenon in AlGaN/GaN HEMT. The electrical and physical properties of these surface traps have been analyzed through the study of 2DEG electron concentration along with the variation of aluminum percentage in the barrier layer of HEMT. This analysis shows that from deep to shallow donors, the percentage change in electron density in 2DEG gets saturated (near 8%) with change in aluminum concentration. The depth of the quantum potential well below the Fermi level is also analyzed and is found to get saturated (near 2%) with aluminum percentage when surface donor states energy changes to deep from shallow. The physics behind this collective effect is also analyzed through band diagram too. The effect of surface donor traps on the surface potential also has been discussed in detail. These surface states are modeled as donor states. Deep donor ( One .  suggeste surface .. [. [. [. [. [Vetury et al.  investig. , 9. Nano. . The fix. . Longoba. [.  performe. [. , 14. Dra. [. [.  have dev[. [. [.  discusse[. [. [. . The rel[. [. [. . Though [. [. [.  used thr[. [. [. . Gucmann[. [. [.  discusseThe above-discussed literature has reported so many relevant aspects of AlGaN/GaN heterostructure but does not account for the combined effect of the surface donor (Concentration + Energy) and contribution of aluminum concentration in the two-dimensional electron concentration. It is evident that aluminum percentage is primarily responsible for polarization charge in AlGaN barrier layer .To provide an appropriate physical understanding of such an effect, we have covered following investigation in the present work (i) the effect on two-dimensional electron concentration with surface donor trap changes from deep to shallow along with aluminum percentage changes in AlGaN barrier layer, (ii) the effect of surface trap and aluminum percentage on surface potential and (iii) the influence of surface donor trap and aluminum percentage on the triangular potential well at AlGaN–GaN interface.2-D device simulations were carried out using Synopsys’s Sentaurus TCAD version L-2016.12 . We caliLG) of 1 μm is placed symmetrically on the top of the AlGaN barrier layer. Un-gated region from gate to drain/source (LGD/LGS) has a dimension of 2.5 μm each and width of the device is 150 μm.The calibrated device has 30 nm AlGaN barrier layer on the top of the 2 μm GaN buffer layer. Schottky gate length  which oversees the semiconductor behavior.PSP is spontaneous polarization, e33 and e31 are piezoelectric coefficients, C33 and C31 are elastic constants, a is the lattice constant and x is a mole percentage of aluminum.The self-consistent drift and diffusion (DD) transport model is also included. Lombardi mobility and Philip unified mobility model were invoked to facilitate mobility degradation due to the electric field and doping. In addition, Auger and SRH  recombination model was used along with Fermi–Dirac statistics. Slotboom model is activated to encounter the bandgap narrowing of heavily doped drain and source extended area. Since this structure has two layers and we are changing the aluminum percentage in the AlGaN barrier layer, the polarization charge is introduced according to the equation of :1\documeσAlGaN (x) with aluminum percentage is depicted in Fig. NC is the effective density of states, F1/2 is Fermi integral of order 1/2, EF is the quasi-Fermi energy for electrons, EC is the conduction band edge and kT represents thermal energy of electrons. Λ is calculated by:ħ = h/2π, h is plank constant, mn is an effective mass of the electron, γ (γ = 1.28) is a fitting parameter and n is electron density.The variation of polarization charge ± σD) on the surface of AlGaN barrier layers Fig. The surface traps were introduced as donor state  of donor trap concentration, there is no significant change in electron density . 2DEG electron density proportionally changes with surface donor concentration (between Region1 to Region2). After a certain threshold value of surface donor trap, again there is no change appearing in the electron density , see Fig. σAlGaN). When the external electric field starts exceeding the internal electric field (due to ± σAlGaN), it brings down the conduction band at the surface and hence contributes more electron to the 2DEG potential well, see Fig. 11 cm−2) for the higher value of donor trap concentration, where 2DEG electron density changes proportionally to the donor trap concentration, Fig. σ) is of the order of 1011 cm−2 for 5% aluminum see Fig. 12 cm−2. So the internal electric field is large enough to pull conduction band offset below Fermi level and hence it forms the 2DEG triangular quantum potential well see Fig. 13 cm−2 for shallow donor traps as shown in Fig. 13 to 3 × 1013 cm−2. Figure (i) The conduction band of the AlGaN barrier layer for region1 has a larger gap from the Fermi level at the surface. As donor trap concentration starts increasing from lower to a higher value, in the transition period (between Region1 to Region2), conduction band proportionally starts moving toward the Fermi level. Thus, 2DEG concentration starts increasing from a lower value to a higher value. In the transition period (between Region1 to Region2) conduction band proportionally starts moving toward the Fermi level and so the donor surface energy also moves toward Fermi level. For Region2, once donor concentration crosses the threshold value, bending of the conduction band starts in such a way that the energy of donor trap pins the Fermi level. Due to Fermi level pining all the donor states get ionized and contribute electrons to the 2DEG triangular quantum potential well. Once the energy of donor states get pinned to the Fermi level, no significant change reflects in the electron density. (ii) To find charge neutrality in the device, surface donor states are essential to counter the electrons in 2DEG. As surface donor states increases, an electric field starts to increase from surface to 2DEG quantum well. This electric field counters the built-in electric field produced by the polarization sheet charge  and 3.6 eV (for surface donor concentration 1 × 1013). This surface potential does not depend on the energy of surface donor trap for its lower value. The surface potential linearly increases as surface donor goes deep (1.4 eV) from shallow (0.2 eV). As surface potential goes down, the 2DEG electron concentration will increase because surface potential linearly varies with surface donor trap energy. Aluminum percentage also have a great impact on the surface potential. Increasing the aluminum percentage from 5 to 50%, electron concentration increases from 7.79 × 1011 to 2.75 × 1013. Surface potential also increases from 0.49 to 0.576 eV when aluminum percentage changes from 5 to 50% see Fig. Some of the literatures have discussed the surface potential variation due to change in aluminum percentage . But thed is the thickness of the AlxGax) eV), where E is the energy below the Fermi level. Two important parameters in the triangular quantum potential well are the depth of potential well below the Fermi level and width of the potential well at the Fermi level. The confinement of the electrons in two-dimension regions is called the 2DEG quantum sheet. The density of states N(E) is one of the important features in the 2DEG quantum potential well. The density of states in two-dimensional quantum well is defined as [m* is the effective mass of the electron and LX is the width of well. This density of state in two-dimensional well looks like a step function.It is important to look at the notch of triangular quantum potential well-formed below the Fermi level  > (E3 − E2)) for higher subbands [EF − E is negative as Fermi level is assumed at zero level, for the deep donor traps (> 0.9 eV to 1.4 eV), which signifies that the energy E is higher than the Fermi level (2DEG is not forming). For shallower surface donor traps (< 0.9 eV to 0.2 eV), the value of EF − E is positive, which means that the value of the E is lower than the Fermi level. For rest of the aluminum concentration (10% to 50%), the value of EF − E is positive which signifies that the value of E is lower than Fermi level and 2DEG notch is forming for all types of surface donor energy (from 0.2 eV to 1.4 eV). It is noted from Fig. E with aluminum gets saturated beyond 20% aluminum concentration, which is also in tune with Fig. EC − 0.2 to EC − 1.4 eV and saturated beyond 20% again. This result is also in tune with Figs. The number of occupied states depends on the Fermi level. For example, if Fermi level is higher than subbands . As subbn 10% to 0%, the vn 10% to 0%, the vIn this paper, we comprehensively present the effect of surface donor traps along with aluminum percentage on electron density and quantum potential well. This manuscript demonstrated that the percentage change happens in 2DEG and notch below the Fermi level gets saturated above 20% of aluminum concentration when surface donor trap energy goes deep to shallow. The electron density in the two-dimensional quantum potential well is saturated approximately at 8%, whereas the energy below the Fermi level saturates somewhere around 2%. These two results are also in tune with each other, except 5% aluminum, having a condition for not forming two-dimensional well for relatively deep (> 0.9 eV) surface donors. Aluminum percentage above 10% forms two-dimensional quantum potential well even for deeper surface donor traps. The effect of surface donor traps on the surface potential also has been discussed in this work. The results of this paper may provide the impetus to the experimental result validation.


Does the cytokine CSF-1 induce monocytes to differentiate into macrophages with a DC-SIGN+ phenotype in endometriosis?Researchers have found that macrophages are the predominant cells in the peritoneal fluid (PF) of endometriosis patients. CSF-1 has been found to accumulate in the lesions and PF of endometriosis patients, and CSF-1 induces THP-1-derived macrophages to polarize toward a CD169+ macrophages were detected by immunohistochemical staining of tissues and flow cytometric analysis of the PF of control subjects (N = 25) and endometriosis (N = 35) patients. The phenotypes and biological activities of CSF-1 -induced macrophages were compared in an in vitro coculture system with peripheral blood lymphocytes from control subjects.The level of CSF-1 in the endometrium of control subjects, and the eutopic, and ectopic endometrium of endometriosis patients was evaluated by real-time polymerase chain reaction (qRT–PCR) and was determined by enzyme-linked immunosorbent assay (ELISA) in the PF of control and endometriosis patients. CSF-1 expression was examined with a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. DC-SIGN+ CD169+ macrophages was higher in the abdominal immune microenvironment of endometriosis patients. CSF-1 was primarily secreted from ectopic lesions and peritoneum in mice with endometriosis. In addition, CSF-1 induced the polarization of macrophages toward a DC-SIGN+ CD169+ phenotype; this effect was abolished by the addition of an anti-CSF-1R antibody. CSF-1 induced the generation of DC-SIGN+ macrophages, leading to a depressed status of peripheral blood lymphocytes, including a high percentage of Treg cells and a low percentage of CD8+ T cells. Similarly, blockade with the anti-CSF-1R antibody abrogated this biological effect.In this study, we found that the proportion of DC-SIGN+ macrophages in the immune microenvironment of endometriosis. Further study of the mechanism and biological activities of CSF-1-induced DC-SIGN+ macrophages will enhance our understanding of the physiology of endometriosis.This is the first study on the role of DC-SIGN Endometriosis (EMS) is a common but complicated disease that causes chronic pelvic pain, dyspareunia and infertility in 6–10% of women of reproductive age , in receA number of studies have been conducted on peritoneal immunity in EMS, but many have been limited to a description of immune cell dysfunction –6. Some + macrophages [+ macrophages are found in various diseases and indirectly affect Treg cells and CD8+ T cells [+ DC-SIGN+ macrophages were found to interact with lymphocytes to maintain transplant survival [+ Treg cells play an important role in immunoregulation and immunosuppression [+ Treg cells secrete cytokines, such as CCL22, CCL17, transforming growth factor-1 (TGF-1), interleukin (IL)-1, tumor necrosis factor (TNF), IL-8 and vascular endothelial growth factor (VEGF), while cooperating with other immune cells to promote the progression of EMS [Peritoneal immunity is complex and powerful, and it is surprising that the ectopic endometrium survives during the menstrual cycle. Colony stimulating factor-1 (CSF-1), also called macrophage colony-stimulating factor (M-CSF), is a key cytokine that induces monocytes to differentiate into CD169rophages . CSF-1 arophages , 16. A srophages . Accordi T cells , 19. In survival . Many stpression , 22. FOXn of EMS .+ T cells are related to EMS [+ macrophages had a profound effect on the cellular immunologic response [+ T cells.Szyllo K, et al. determined that a small percentage of CD8d to EMS . A studyresponse . Moreoveresponse . Therefo+ macrophages is a key target in the regulation of depressed immunity. This molecule was first described by Geijtenbeek TBH, et al., in a study about a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells [+ T cells [+ DCs regulated CD8+ T cells [+ DC-SIGN+ phenotype in endometriosis and that this macrophage phenotype can influence the differentiation of T cells.DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN or CD209) on CD169 T cells . Mechani T cells . Soilleu T cells . Marie L T cells . Recentl T cells . Overall+ macrophages were detected by immunohistochemical staining of tissues and flow cytometric analysis of the PF of control subjects and endometriosis patients. The phenotypes and biological activities of CSF-1 -induced DC-SIGN+ macrophages were compared using flow cytometric analysis in an in vitro coculture system with peripheral blood lymphocytes.CSF-1 was evaluated by qRT–PCR in the tissues of control subjects and endometriosis patients, and was determined by ELISA in the PF of control and endometriosis patients. Then, CSF-1 expression was examined with a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel in the EMS model. DC-SIGNn = 30) or Stage IV (n = 9)) who were diagnosed by clinical pathologists at Shanghai First Maternity and Infant Hospital Affiliated with Tongji University School of Medicine, between June 2019 and May 2020 were recruited. Eutopic endometrial samples were obtained from women with EMS (n = 17). For the control samples, endometrial tissue samples were collected by curettage from 11 women who exhibited fallopian tube blockage and previously needed contraception by fallopian tube ligation but now needed tubal reversal, and did not have any clinical indication or history of adenomyosis or endometriosis. PF was collected, avoiding contamination with blood, from women with advanced-stage EMS (n = 35) and from control subjects (n = 25) during laparoscopic surgery. All the enrolled patients in the control group were confirmed by laparoscopy to have no endometriotic lesions, and all had a histological diagnosis of mature ovarian teratoma composed of epidermis only (n = 20) or benign cyst of the mesosalpinx (n = 5). The clinical characteristics of the patients and controls are summarized in Table For immunohistochemical staining, 39 women with EMS (American Fertility Society classification as endometriosis Stage III ( The acute monocytic leukaemia (THP-1) cell line was a gift from the Cryomedicine Lab at Qilu Hospital of Shandong University. Recombinant human M-CSF was purchased from R&D Systems . C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., at 4 weeks of age.N = 11) and the eutopic endometrium (N = 17) and ectopic (N = 39) endometrium of endometriosis patients, we conducted PCR. Total RNA was extracted from 0.2 × 0.2 × 0.1 cm tissue fragments using TRIzol Reagent  according to the product manual. RNA (0.5 μg) was reverse transcribed into cDNA with ReverTra Ace Quantitative PCR (qPCR) RT Master Mix with gDNA Remover . Each 20 μl PCR mixture contained 1 × SYBR Green PCR Master Mix , 30 ng of cDNA and 300 nM each specific primer from Sangon Biotech (Shanghai) Co., Ltd. Quantitatie PCR was performed on an Applied Biosystems StepOnePlus RT–PCR System . Three separate experiments were performed on different cultures, and each sample was assayed in triplicate. The mean was calculated to determine the mRNA levels by quantitative real-time polymerase chain reaction (RT–PCR) analysis using the ABI StepOnePlus RT–PCR System (Applied Biosystems). The primers used to amplify CSF-1 were 5'-GCTGCTTCACCAAGGATTATG-3' (sense) and 5'-GGGTCACTGCTAGGGATG-3' (antisense), and the gene expression levels in each group was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was assessed with the following primers: 5'-AGCCACATCGCTCAGACA-3' (sense) and 5'-GCCCAATACGACCAAATCC-3' (antisense). The mean relative gene expression levels were determined, and differences in expression were calculated using the 2−△△Ct method.To test the gene expression level of CSF-1 in the endometrium of control subjects (All mice were maintained under specific pathogen-free (SPF) conditions. All procedures were approved by the Animal Care and Use Committee of Tongji University . The estrous stage was monitored daily by obtaining a vaginal smear every morning for 2 weeks, and mice with a normal estrous cycle were used in the following experiments. Endometriosis was established by intraperitoneal injection of endometrial segments. Briefly, donor mice were sacrificed, and the uteri were isolated and collected in a Petri dish containing phosphate-buffered saline (PBS). The isolated uterine horns were treated identically, including longitudinal splitting with a pair of scissors, isolation of endometrial tissue and gentle disruption to generate small, uniform fragments of approximately 1 mm. Then, these fragments were intraperitoneally injected into recipient mice through a 1 ml syringe with a 25 G needle. Equal amounts of endometrial fragments from one donor mouse were injected into two recipient mice. Specifically, 15 endometrial segments in 200 μl of sterile PBS were injected into each recipient mouse. However, mice in the control groups only received injections of 200 µL of sterile PBS. Finally, ten mice from the endometriosis and control groups were sacrificed 42 days after the model was established.A MILLIPLEX® MAP kit for analysis was used to evaluate tissue  cytokine production (CSF-1) and release in the PF of EMS mice, in accordance with the manufacturer’s instructions. In addition, the plate was analyzed on a Luminex 200 system. The sample data were analyzed using xPONENTLX200 software .+, CD169+, and CSF-1R+ macrophages and FOXP3+ T cells in the tissues of control subjects (N = 25) and endometriosis patients (N = 35), we conducted immunohistochemical staining. For each sample, 4 μm thick sections were dewaxed and rehydrated in ethanol and water. Antigen retrieval was performed in citrate buffer , and endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide. The tissue sections were incubated overnight at 4 °C with rabbit primary antibodies against human CD169 , CSF-1R , and DC-SIGN  and mouse monoclonal antibodies against human CD68  and FOXP3 . A secondary antibody kit  was utilized to label the primary antibodies. The sections were counterstained with hematoxylin, dehydrated in ethanol and xylene, and mounted in PermountTM mounting medium. Images were acquired using a microscope , which was fitted with a digital camera . A series of five images was randomly selected from six sections per tissue sample. Positive cells in images acquired at 400 × magnification were counted manually by two independent investigators blinded to the treatment groups. Cells were counted in at least five different areas in ectopic lesions, and data were plotted as the percentage of positive cells relative to the total number of cells, as previously described [To detect DC-SIGNescribed . The perTHP-1 cells were induced to differentiate into macrophages in accordance with a previously reported method . Cells c+ and CD169+ macrophages derived from THP-1 cells induced by CSF-1 and blocked with CSF-1R, we conducted a cellular immunofluorescence analysis. We treated the differentiated macrophages with CSF-1 (50 ng/ml) and cultured them for 48 h. For confirmation, we cultured the differentiated macrophages with RPMI 1640 medium  containing anti-CSF-1R  for 2 h and then added CSF-1  to the medium for 48 h. Cellular immunofluorescence staining was performed as previously described [To detect the DC-SIGNescribed . The tar+ CD169+ macrophages derived from THP-1 cells on T cells, we cocultured PBMCs with THP-1-derived macrophages. PBMCs were prepared as previously described [n = 7) and with a regular menstrual cycle. PBMCs were isolated from 15 ml of venous blood using Ficoll-Cardiografin . After centrifugation, PBMCs were collected from the interphase layer and washed four times with PBS. For coculture, PBMCs were suspended in RPMI 1640 medium supplemented with FBS at a concentration of 1 × 106 cells/ml. The coculture system simulated the lesion site in the human body. Transwell membranes  were used to allow the exchange of macrophage secretions with PBMCs. First, Transwell membranes were inserted into a 24-well plate, and the upper chamber was preseeded with macrophages derived from THP-1 cells. In addition, 100,000 PBMCs were counted and seeded on the membrane. Second, two groups of macrophages derived from THP-1 cells were treated with a rabbit anti-human DC-SIGN  or CSF-1R  antibody and with CSF-1 (50 ng/ml) for 48 h. Third, we cocultured the differentiated macrophages with PBMCs, as shown in , FITC-anti-human CD45 , PerCP-Cy5.5-anti-human CSF-1R  and PE-anti-human CD169 . Fluorophore-conjugated and isotype-matched antibodies served as the negative controls. The proportions of Treg cells and CD8+ T cells in the PBMCs collected from the coculture system were determined using FCM. PBMCs at a density of 0.5 × 105 cells/tube were fixed, permeabilized, and stained in duplicate with the following antibodies: APC-anti-human FoxP3 , FITC-anti-human CD45 , PerCP-Cy5.5-anti-human CD8 , and PE-anti-human CD4 . The proportions of distinct subpopulations were eventually characterized on a BD FACSCalibur . The resulting data were examined using FlowJo software. A representative graph from three or more repeated experiments is shown. Positive gates were set separately for each sample type and label and were set to include less than 1% of the acquired events in the unstained sample.The remaining peritoneal cells collected from the controls and the patients were divided into two equal parts for analysis by flow cytometry (FCM). To determine the percentages of the DC-SIGNFirst, an aliquot of each preparation was kept for protein estimation using a Bradford protein concentration assay kit (P0006C) from Beyotime. The CSF-1 levels in the PF of EMS patients and controls were quantified using each ELISA kit (R&D Systems), according to the manufacturer’s instructions. The absorbance of the streptavidin–horseradish peroxidase colorimetric reaction product was measured with a microplate reader capable of measuring the absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. The optical density values were compared with those of serial dilutions of recombinant human CSF-1 as a standard. The relevant concentration was calculated, and standard curves were generated.P < 0.05 was considered to indicate significance.All statistical analyses were completed using Prism software, version 5 , and the measurement data are presented as the mean ± SD values. Statistical significance was assessed using Student’s t test for univariate analyses of two sets of data by one-way or two-way analysis of variance (ANOVA) when multiple comparisons were necessary. We collected peritoneal cells from the PF of patients for flow cytometry and performed a series of analyses Fig. . Macroph+ macrophages and DC-SIGN+ macrophages than in the normal endometrium and in the eutopic endometrium of endometriosis patients. Additionally, in ovarian endometriotic lesions, we observed significantly higher numbers of FOXP3+ T cells than in the normal endometrium or in the eutopic endometrium of the endometriosis patients. In contrast, concerning CD169+ macrophages and CSF-1R+ macrophages, there were no significant differences between the ovarian endometriotic lesions, the normal endometrium and the eutopic endometrium of the endometriosis patients (Fig. We also conducted immunohistochemical staining and performed a series of analyses of the immune cells detected in all the tissues Fig. A. In ovants Fig. B.Fig. 2MWe observed a relative increase in CSF-1 gene expression in ectopic endometrial tissue compared with the paired eutopic endometrial tissue and normal endometrial tissue Fig. A. Our da+ macrophages was observed in the CSF-1-treated group, while in this model, immunofluorescence staining showed that, after the CSF-1 antagonist anti-CSF-1R was added, the expression of CD169 was low (Fig. The successful induction of macrophages from THP-1 cells was monitored by evaluating the characteristic CD68-positive cells Fig. . A signilow Fig. A. Similalow Fig. B.Fig. 4C+ macrophages induced by CSF-1 led to a depressed phenotype of peripheral blood lymphocytes, while the CSF-1 antagonist anti-CSF-1R abrogated this biological effect (Fig. On the basis of the successful induction of macrophage differentiation from THP-1 cells, we established a coculture system with macrophages and peripheral blood lymphocytes Fig. A. Lymphoect Fig. C.Fig. 5C+ macrophages [+ macrophages; similarly, anti-CSF-1R abolished this effect. Moreover, we found that DC-SIGN+ macrophages led to a suppressive phenotype of peripheral blood lymphocytes, including a high percentage of Treg cells and a low percentage of CD8+ T cells, which was agreement with previous study [CSF-1 is a cytokine that induces monocytes to differentiate into CD169rophages . Our stuus study ; and thi+ macrophages, which are known as CD169+ suppressive macrophages [+ CD169+ macrophages in the pathogenesis of endometriosis has not been studied. We found that the proportion of CD169+ DC-SIGN+ macrophages was higher in the PF of endometriosis patients; this phenomenon was not in agreement with observations in tissues. Although DC-SIGN+ macrophages were more abundant in the ectopic endometrium group than in the eutopic endometrium and the normal endometrium groups, the number of CD169+ macrophages was not different among the three groups. These differences may be due to the diversity of phenotypic differentiation of macrophages in different tissues and regions.DC-SIGN is expressed in human DCs and macrophages , 37. Improphages , play a We also found that macrophages coexpressing CSF-1R and CD169 were more abundant in the PF of endometriosis patients than in that of controls. A previous study proved that CSF-1R inhibition altered macrophage polarization and blocked glioma progression . CSF-1R + DC-SIGN+ suppressive macrophages.Although the protein expression level of CSF-1R in the ectopic lesions of endometriosis patients was not different from that in the eutopic endometrium of endometriosis patients and the normal endometrium, the gene expression level of CSF-1 was significantly higher in these lesions. The level of the CSF-1 cytokine in the PF of endometriosis patients was higher than that in the PF of controls. Our in vivo experiment showed that the peritoneum of all mice contained high levels of the cytokine CSF-1, while the level of the cytokine CSF-1 was significantly higher in the ectopic endometrial tissue of mice with endometriosis than in the eutopic endometrial tissue of mice with endometriosis and the normal endometrium of control mice. The principal source of CSF-1 may be ectopic lesions in mice with endometriosis. This result was in agreement with a previous study that proved that compared with eutopic tissue, ectopic endometrial tissue exhibited a 3.5-fold increase in CSF-1 expression . Therefo+ DC-SIGN+ macrophages. We also demonstrate here that CSF-1-induced DC-SIGN+ macrophages inhibit the increase in CD8+ T cells and the decrease in Treg cells. We are studying the detailed mechanism by which DC-SIGN+ macrophages affect the proliferation and differentiation of T cells in EMS. This study provides a framework for developing CSF-1-based in vitro protocols to generate therapeutic macrophages for clinical use to regulate the immune environment of endometriosis.Overall, CSF-1 and its receptor, CSF-1R, are important for the induction of CD169


Acetazolamide reduced CSF secretion by ~50% across all groups, n = 7, t(6) = 4.294, p = 0.005. Conclusions: Acute antagonism of NKCC1 and TRPV4 proteins at the choroid plexus does not reduce CSF secretion in healthy rats. Further investigation of protein changes and antagonism should be explored in neurological disease where increased CSF secretion and ICP are observed before discounting the therapeutic potential of protein antagonism at these sites.Background: Cerebrospinal fluid (CSF) secretion can be targeted to reduce elevated intracranial pressure (ICP). Sodium-potassium-chloride cotransporter 1 (NKCC1) antagonism is used clinically. However, supporting evidence is limited. The transient receptor potential vanilloid-4 (TRPV4) channel may also regulate CSF secretion and ICP elevation. We investigated whether antagonism of these proteins reduces CSF secretion. Methods: We quantified CSF secretion rates in male Wistar rats. The cerebral aqueduct was blocked with viscous mineral oil, and a lateral ventricle was cannulated. Secretion rate was measured at baseline and after antagonist administration. Acetazolamide was administered as a positive control to confirm changes in CSF secretion rates. Results: Neither NKCC1, nor TRPV4 antagonism altered CSF secretion rate from baseline,  Intracranial pressure (ICP) elevation is reported in several neurological conditions including hydrocephalus ,2, idiop3− accumulation and has been shown to rapidly decrease ICP in healthy rats [+ and Cl− are the predominant ions present in CSF [+, K+, and Cl− is an important driver of CSF secretion. The resulting osmotic gradient allows passive water transport into the ventricles via the aquaporin 1 (AQP1) water transporter [Pharmaceuticals for managing ICP are limited. Acetazolamide reduces CSF secretion by ~50% by inhibiting carbonic anhydrase, an intracellular enzyme in the choroid plexus epithelia contributing to HCOthy rats ,20. Acetthy rats . It is othy rats . Howeverthy rats . Furtherthy rats ,25,26. Tthy rats ,28,29,30t in CSF . NKCC1 insporter . AQPs arnsporter . AQP1, 4nsporter . Clinicansporter ,37,38,39nsporter . Anothernsporter . Furosemnsporter . These snsporter . This tensporter ,43. TherThe transient receptor potential vanillioid-4 (TRPV4) channel is highly expressed at the apical membrane of the choroid plexus and is also highly expressed in renal tissue ,45. ActiWe aimed to determine whether antagonism of NKCC1 or TRPV4 proteins reduces CSF secretion acutely in healthy rats. The intention of this study was to build an evidence base to support the role of NKCC1 and TRPV4 as targets for managing CSF secretion before investigating their therapeutic potential for managing ICP in neurological disease and injury. We anticipated that antagonism of NKCC1 would reduce CSF secretion due to the existing clinical applications of loop diuretics and some pre-clinical studies that support the use of bumetanide for managing ICP. We suspected that TRPV4 antagonism would also result in reduced CSF secretion due to its success in ameliorating symptoms of CSF accumulation in hydrocephalus. Antagonism of NKCC1 nor TRPV4 reduced CSF secretion rate in healthy rats, suggesting that neither protein is likely to act as a sole regulator of CSF secretion under normal conditions. However, we cannot discount these proteins as targets of CSF secretion and ICP rise in neurological disease or injury. Further work must explore responses to antagonism under pathological conditions.The experimental timeline is outlined in The TRPV4 antagonist, RN1734, in dimethylsulfoxide (DMSO), was administered intraperitoneally as previously described . Althougn = 17). All experimental animal procedures used in this project were approved by the Animal Care and Ethics Committee of the University of Newcastle (A-2013-343 & A-2020-003).Procedures were carried out on male outbred Wistar rats aged between 12–16 weeks and weighing between 265–330 g . Core body temperature was regulated via a thermocoupled rectal probe  and heat mat.Rats were anaesthetised with isoflurane  in 50:50% NA catheter was inserted into the saphenous branch of the femoral artery and was used to obtain mean arterial blood pressure and heart rate. Blood samples (0.1 mL) were taken from the catheter for arterial blood gas and pH measurements using a fast blood analyser . A similar catheter was placed into the femoral vein for infusion of acetazolamide. Oxygen saturation and respiration were monitored throughout procedures.CSF secretion was measured as described in Karimy et al. . AnimalsTo expose the cisterna magna, the back of the neck was shaved, and a 1 cm incision made in the space between the occipital bone and the axis (C1 vertebrae). The suboccipital muscles were dissected from the occipital bones to expose the atlanto-occipital ligament. The neck was then rotated 90° on the ear bars so that its nose was pointing down. The ligament was resected to expose the underlying dura. The dura was then punctured (1 mm diameter) to access the cisterna magna. A polyethylene catheter  loaded with sterile, molecular grade mineral oil was inserted through the membrane and advanced 5 mm through the foramen of Magendie into the fourth ventricle. Mineral oil (100 μL) was infused into the fourth ventricle to occlude the aqueduct of Sylvius. The catheter was sealed into the cisterna magna using a sealant to prevent movement and unblocking of the aqueduct.The left lateral ventricle was then cannulated using a 27 G neonatal lumbar needle  attached to silastic tubing . The tubing began to fill with CSF upon cannulation and the distance (millimetres) travelled at 5 min intervals was recorded. We used the following equation to calculate the volume of CSF secreted:The rate of CSF secretion (μL/min) was calculated as the slope of the volume-time relationship.n = 4), 10 mg/kg NKCC1 antagonist bumetanide in ethanol , or 1 mL/kg DMSO intraperitoneally (n = 3). After the three-hour testing period was complete, seven animals received 10 mg/kg acetazolamide in saline (0.9%) intravenously as a positive control for reduced CSF secretion.Animals received either 10 mg/kg of the TRPV4 antagonist RN1734 in dimethyl sulfoxide  .Statistical analyses were carried out using GraphPad Prism 7 . Comparisons between pre-drug and post-drug physiological variables were conducted for each treatment group using the paired Student’s Six animals were excluded from the study due to absence of CSF flow from the cannulated ventricle into the ventricular catheter and tubing. One animal died prior to drug administration. All exclusions were prior to group assignment. Ten animals were included in the final study.t-test).Physiological parameters of each treatment group are presented in n = 7, t(6) = 4.29, p = 0.005.Acetazolamide reduced CSF secretion from 0.34 ± 0.08 µL/min at baseline to 0.17 ± 0.05 µL/min post-infusion when averaged across all groups, n = 3, t(2) = 1.14, p = 0.37; n = 2, t(1) = 1.73, p = 0.33; Bumetanide did not change CSF secretion rate  = 0.58, p = 0.6, n = 3, t(2) = 7.07, p ≤ 0.02; The average CSF secretion rate did not change after RN1734 was administered  = 0.47, p = 0.69; n = 2, t(1) = 5.32, p = 0.12; DMSO did not alter the CSF secretion rate (In this study, we explored the potential of targeting NKCC1 and TRPV4 to control CSF secretion. Pharmacological therapies like these would be advantageous in neurological diseases where increased CSF secretion contributes to increased ICP. We used a direct approach for measuring CSF secretion and found that antagonism of NKCC1 and TRPV4 did not decrease CSF secretion rates in healthy rats naïve to neurological disease or injury. This suggests that these proteins are ineffective targets for controlling CSF secretion, at least in the absence of neuropathological mechanisms. Bumetanide and other loop diuretics acting on NKCC1, like furosemide, were previously used for the clinical management of elevated ICP ,25. HoweWe hypothesised that TRPV4 antagonism would reduce CSF secretion, as previous studies of TRPV4 expression sites and ion transport activity have alluded to a regulatory role for the protein in CSF secretion ,63. AddiOur direct approach for measuring CSF secretion was advantageous over tracer dilution techniques for measuring CSF secretion as values are less likely to be overestimated due to tissue uptake of tracer ,65. The We hypothesised that NKCC1 or TRPV4 antagonism would reduce CSF secretion rates in rats, as both proteins have been implicated in CSF secretion and have been targeted for mitigating ICP elevation and symptoms of increased cranial CSF volume. We conclude that acute antagonism of either NKCC1 or TRPV4 is ineffective at reducing CSF secretion in rats absent of neurological disease or injury. Further investigation of protein changes and antagonism could be explored in neurological disease where increased CSF secretion and ICP are observed before discounting the therapeutic potential of protein antagonism at these sites.


Ischemic heart disease is a leading cause of mortality and morbidity worldwide. We previously demonstrated that acacetin protects against myocardial ischemia reperfusion injury in rats, although the underlying mechanism remains to be elucidated. In the present study, we investigated the effects of acacetin on autophagy during hypoxia/reoxygenation (H/R) injury by exposing H9c2 myocardial cells to H/R with or without acacetin pretreatment during hypoxia. Our results show that acacetin significantly increased cell viability in a dose-dependent manner, enhanced antioxidant capacity, and suppressed protein apoptosis of rat cardiomyocytes H9c2 cells following H/R injury. In addition, lentiviral infection of H9c2 cardiomyocytes revealed that acacetin pretreatment significantly enhanced the fluorescence intensity of autophagy proteins Beclin 1, LC3-II, and p62. These results indicate that acacetin protected H9c2 cardiomyocytes from H/R damage by enhancing autophagy. Moreover, we found that application of acacetin increased activation of the PI3K/Akt signaling pathway, whereas cotreatment with the PI3K inhibitor LY294002 reversed the inhibition of apoptosis and autophagy induced by acacetin. In conclusion, acacetin mitigated H/R injury by promoting autophagy through activating the PI3K/Akt/mTOR signaling pathway. Ischemic heart disease is a leading cause of mortality and morbidity worldwide . FollowiZiziphora bungeana (Juz.), was previously demonstrated to protect against myocardial ischemia reperfusion injury , 3332, 3In summary, the results of this study suggest that acacetin inhibited apoptosis through the PI3K/Akt/mTOR signaling pathway to alleviate H/R injury in H9c2 cardiomyocytes by promoting autophagy. Thus, the clinical efficacy and safety of acacetin for reducing myocardial H/R injury warrant further study.


Although endometriosis is a benign disease characterized by the presence of endometrial tissues outside the uterus, ectopic endometrial cells can exhibit malignant biological behaviors. Retinol-binding protein4 (RBP4) is a novel adipocyte-derived cytokine, which has important roles in regulating insulin sensitivity and energy metabolism. RBP4 is a potent modulator of gene transcription, and acts by directly controlling cell growth, invasiveness, proliferation and differentiation. Here, we evaluated the possible role of RBP4 in the pathogenesis of endometriosis. We compared the levels of RBP4 in the tissues and peritoneal fluid (PF) of women with and without endometriosis and evaluated the in vitro effects of RBP4 on the viability, invasiveness, and proliferation of endometrial stromal cells (ESCs). RBP4 levels were significantly higher in the PF of the women in the endometriosis group than in the controls. RBP4 immunoreactivity was significantly higher in the ovarian endometriomas of women with advanced stage endometriosis than those of controls. In vitro treatment with human recombinant-RBP4 significantly increased the viability, bromodeoxyuridine expression, and invasiveness of ESCs. Transfection with RBP4 siRNA significantly reduced ESC viability and invasiveness. These findings suggest that RBP4 partakes in the pathogenesis of endometriosis by increasing the viability, proliferation and invasion of endometrial cells.  Endometriosis is a common chronic inflammatory disease that is characterized by the presence of endometrial glands and stroma outside the uterine cavity. It affects 5–10% of the women in the reproductive age and affects up to 80% of the women with pelvic pain, and 20–50% of the women with infertility . This thAdipokine (or adipocytokine) is a family of cytokine that is secreted mainly from adipose tissue . AlthougThe adipokine (or adipocytokine) family comprises cytokines that are primarily secreted from adipose tissues . AlthougRetinol-binding protein 4 (RBP4) is a plasma retinol transporter that carries retinol from the liver to the periphery, and similar to adipokines, plasma RBP4 originates from adipose tissues . RBP4 isBased on the emerging importance of various adipokines in endometriosis, it follows that the expression of RBP4 may have a potential role in the pathogenesis of endometriosis, which is characterized by the cell growth, invasion, differentiation, migration, and activation of cytokines, chemokines, and growth factors. However, the role of RBP4 in endometriosis has not been investigated to date. Therefore, we aimed to evaluate the possible role of RBP4 in the pathogenesis of endometriosis in this study. We compared the levels of RBP4 in the peritoneal fluid (PF) and tissues of women with and without endometriosis. We also evaluated the in vitro effects of RBP4 on the viability, proliferation, and invasiveness of endometrial stromal cells (ESCs), and investigated the functional alterations in ESCs induced by the inhibition of RBP4.n = 6) compared to controls (n = 6), we compared the expression levels of 105 cytokines, chemokines, and growth factors between the two groups utilizing a human XL proteome profile array . The expression levels of angiogenin, cluster of differentiation 105 (CD105), inter-cellular adhesion molecule 1 (ICAM-1), chemokine (C-X-C motif) ligand 10 (CXCL10), leptin, lipocalin-2, matrix metallopeptidase 9 (MMP-9), osteopontin, RBP4, plasminogen activator inhibitor-1 (PAI-1), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor binding protein (ABP), CD31, thermal interface material 2 (TIM-2) and vascular cell adhesion molecule 1 (VCAM-1) were significantly higher in the endometriosis group compared with the control group . Among those soluble proteins significantly increased in PF in patients with endometriosis compared with controls, we found that the increase in the levels of RBP4 in endometriosis has not been reported to date.In order to identify which cytokines, chemokines, or growth factors are elevated in PF in patients with endometriosis (n = 6) and without (n = 6) endometriosis, and in the fluids from ovarian endometriomas (n = 6) using ELISA. The levels of RBP4 in the PF were significantly higher in the endometriosis group compared with the control group (P < 0.05) (P <0.05) and the control group (P < 0.05)  C. The le < 0.05) C.p < 0.001; p < 0.001, respectively), the proliferative phase , and the secretory phase  (p < 0.01), and the secretory phase (p = 0.002) (p = 0.012)  B, left. = 0.002) B, right.= 0.012) B, right.p < 0.05) or transfected with the negative control siRNA (p < 0.05) (p < 0.05)  or transfected with the negative control siRNA (p < 0.05) (p < 0.05)  or transfected with the negative control siRNA (p < 0.05) after 48 h   (p < 0.05) (p < 0.05)  to those of all the cells. The population of BrdU-positive cells significantly decreased after 48 h of transfection with RBP4 siRNA (3.5%)  gene, was shown to be markedly elevated in the serum and PF of patients with endometriosis , and it Given that the prevalence of endometriosis is inversely associated with body mass index (BMI) or obesity , the cauConsidering that RBP4 serves as a signaling molecule activating downstream pro-oncogenic signaling pathways after binding to the membrane receptor STRA6 , the datIn this study, it was not possible to clarify whether the increased RBP4 level or expression itself can play a crucial role in the establishment of endometriosis or may have resulted from disease progression due to the activation of pro-inflammatory processes, endometrial proliferation, or invasion after the initial establishment of endometriotic lesions. All the women in the control group were not disease-free, and had benign gynecological diseases other than endometriosis. We could not explain the main origin of the increased of RBP4 levels in the patients with endometriosis, which is well known to be inversely associated with BMI or fat mass. Despite these limitations, to our knowledge, this study is the first to demonstrate a marked elevation in the levels of RBP4 in the PF of women with endometriosis as well as a significant increase in the expression of RBP4 in ovarian endometriomas, and to elucidate the functional relevance of RBP4 in the development of endometriosis. Further well-designed study is necessary to overcome the limitations of the present study.n = 6) and from the control group (n = 6) during laparoscopic surgery. The PF was collected from the posterior cul-de-sac or utero-vesical pouch via a laparoscopic cannula. The samples of PF with blood contamination were excluded. The fluids from the ovarian endometrioma were also obtained from patients with advanced stage endometriosis (n = 6) during laparoscopic surgery using a laparoscopic needle. The cellular components of the PF and endometriotic fluids were removed by centrifugation at 3500 revolutions per minute for 15 min. The supernatants were subsequently collected and stored in aliquots at −70 to −80 °C until further analyses. The PF was collected in the follicular phase from patients with advanced stage endometriosis (2) than in the endometriosis–hysterectomy group  and the ovarian endometrioma group . For immunohistochemical staining, we used the sections of endometria and ovarian endometriomas from our previous study . BrieflyFor the ESC cultures, endometrial samples were obtained from fertile women diagnosed with intramural leiomyoma at the time of hysterectomy, who had no evidence of endometrial abnormalities, adenomyosis, or pelvic endometriosis, and who had not received any hormonal medications in the preceding 3 months. The endometrial samples were placed in Hanks’ balanced salt solution and transported to the laboratory for the isolation and culture of ESCs. Written informed consent was obtained from each patient using consent forms and protocols approved by the Institutional Review Board for Human Research of Asan Medical Center. n = 6 per group) was performed separately in duplicate, and the average of these two-pixel densities was used to calculate the average density using Image J software. The background staining and spot sizes were analyzed according to the manufacturer’s instructions. The images were converted to 8-bit inverted jpeg files and the spots were encircled. Equal spot sizes were analyzed for each blot.A human XL proteome profile array was initially performed for analyzing the PF, according to manufacturer’s instructions , for identifying the cytokines, chemokines, and growth factors that are elevated in the PF of patients with endometriosis, in comparison to that of the control. The blotting of the PF samples . Deparaffinization, epitope retrieval, and immunostaining were performed according to the manufacturer’s instructions by using cell-conditioning solutions (CC1) and the BMK ultraVIEW diaminobenzidine detection system . The sections of the lesions were stained with RBP4 . The positive signals were amplified using ultra-VIEW copper, and the sections were counterstained with hematoxylin and DAB. Images of the slides were captured using a Vectr intelligent Slide Analysis System . The images were subsequently used to train the Form Advanced Image Analysis Software  for quantitative image analysis.The ESCs were separated and maintained in a monolayer culture as previously described . FollowiEach of the experiments on the ESCs was performed using cells separately prepared from the endometrial tissue specimens obtained from different patients. When the culture had reached a confluence of 70%, the ESCs were treated with serum-free, phenol red-free media  for 48 h before treatment. In order to evaluate the effect of HR-RBP4  and RBP4 siRNA on cellular viability, invasiveness, the expression of MMP-2/9, and proliferation, the cultures were separately treated with the vehicle (control), 25 ng/mL HR-RBP4, 30 nmol/L negative control siRNA, or 30 nmol/L RBP4 siRNA for 48 h.n = 4). The cells were subsequently detached and seeded on 96-well plates at a density of 5000 cells/well. Then, 10 μL of the highly sensitive, water soluble tetrazolium salt assay reagent was added to each well on day 2 for the ESCs. The plates were incubated for 1 h at 37 °C. Cell viability was determined by measuring the absorbance of the samples at 450 nm.Cell viability was assessed using an EZ-Cytox cell viability assay kit . The ESCs transfected with RBP4 siRNA or the negative control siRNA were incubated for 48 h (n = 4). The cells were then starved in serum-free DMEM for 24 h. Then, 50 μL of cell suspension  was added to the top compartment and 100 μL of serum-free DMEM was added to the bottom compartment of the chamber. The chambers were incubated at 37 °C in humidified air containing 5% CO2 for 24 h. Both the compartments of the chambers were subsequently washed with washing buffer, and the cells that invaded the bottom chamber were labeled by incubating with 5 μg/mL calcein-AM  in cell dissociation solution for 1 h at 37 °C. Cell invasion was determined by measuring the absorbance of the samples at excitation and emission wavelengths of 485 nm and 520 nm, respectively, with the same parameters (time and gain), using a Victor X3 multilabel plate reader (PerkinElmer Inc.).Invasion assays were performed in a 96-well trans-well containing inserts with a pore size of 8 μm , and coated with Cultrex Basement Membrane Extract . The ESCs transfected with RBP4 siRNA or the negative control siRNA were incubated for 48 h (n = 4). The membranes were blocked by incubation for 1 h at room temperature, and they were subsequently incubated over-night with the primary antibodies against MMP-2 , MMP-9 , and actin  at 4 °C. The membranes were washed thrice and subsequently incubated with a secondary horseradish peroxidase-conjugated anti-IgG antibody  and visualized using a Pierce chemiluminescent substrate . The densitometric quantification of the bands was performed using a laser densitometer. The bars represent the mean of data obtained from three replicates.The cells were resuspended in lysis buffer  containing a protease inhibitor cocktail  (5 cells/well for 24 h and treated with HR-RBP4 and the negative control siRNA for 48 h (n = 4). The cellular proliferation in these cultures was measured by flow cytometry  using a Cell Proliferation Kit , according to the manufacturer’s protocol. The assays were performed in triplicate and the results represent the data obtained from three independent experiments.The ESCs were seeded onto 6-well culture plates at a density of 2 × 10P < 0.05 was considered to be statistically significant. All the data were analyzed by the Kolmogorov–Smirnov test to evaluate whether they followed a normal distribution. When the data followed a normal distribution, continuous variables were compared using Student’s t test (two groups) or analysis of variance and Fisher’s least significant difference post hoc test for pairwise comparisons (three groups). The Mann–Whitney U test was used for comparing the data (two groups) when the data did not follow a normal distribution. All the statistical computations were performed using the Statistical Program for the Social Sciences software, version 14.0. The results of this study strongly suggest that RBP4 might partake in the pathogenesis and/or pathophysiology of endometriosis by increasing the viability, proliferation, and invasiveness of endometrial cells.


Purpose: Cerebral aspergillosis (CA) is a rare but often fatal, difficult-to-diagnose, opportunistic infection. The utility of metagenomic next-generation sequencing (mNGS) for diagnosis of CA is unclear. We evaluated the usefulness of mNGS of the cerebrospinal fluid (CSF) for the diagnosis of CA.Methods: This prospective study involved seven consecutive patients with confirmed CA in whom CSF mNGS was performed. Serum (1→3)-β-D-glucan and galactomannan levels were determined, and histopathological examination and mNGS of the CSF were conducted. CSF specimens from three non-infected patients were used as positive controls.Results: mNGS of the CSF was positive in six of the seven confirmed CA cases (85.71% sensitivity). In the cryptococcal meningitis group (control), mNGS of the CSF was positive for Aspergillus in two patients (84.62% specificity). The positive likelihood ratio, negative likelihood ratio, and Youden’s index of mNGS for CA in the CSF were 5.565, 0.169, and 0.7, respectively. Among the six mNGS-positive cases, more than two Aspergillus species were found in four . In the positive controls, the addition of one A. fumigatus spore yielded a standardised species-specific read number (SDSSRN) of 25.45 by mNGS; the detection rate would be 0.98 if SDSSRN was 2.Conclusion: mNGS facilitates the diagnosis of CA and may reduce the need for cerebral biopsy in patients with suspected CA.Trial Registration Number: Chinese Clinical Trial Registry, ChiCTR1800020442. Aspergillus species. This notorious complication of invasive aspergillosis, which accounts for 5–10% of all intracranial fungal pathologies  is a rare and life-threatening opportunistic infection caused by hologies , is assohologies . The majhologies . CA typihologies . The golhologies . Howeverhologies . Therefohologies .Aspergillus, which is widespread in the environment (Metagenomic next-generation sequencing (mNGS) enables diagnosis of infectious diseases of the central nervous system (CNS)  and is iironment , and assWe prospectively identified seven consecutive patients with confirmed CA admitted to two teaching hospitals in Beijing, China between November 2016 and September 2019. We recorded the patients’ clinical data, including relevant medical history, physical examination findings, routine blood examinations, CSF parameters, and neuroimaging findings. Diagnosis of CA was confirmed by CSF culture or histopathological evidence. Because of the similar risk factors for CA and cryptococcal meningitis (CM), 13 patients with CM confirmed by CSF India ink smear and/or culture were used as controls.Aspergillus spores (A. fumigatus B5233 wild type) were added to sterile CSF for sensitivity testing of mNGS in spiked specimens and as positive controls. The number of A. fumigatus spores in the three 0.6-mL amounts of CSF was 50, 250, and 500.Sterile CSF was collected from three non-infected patients who required lumbar puncture and divided into three 0.6-mL tubes. Next, different concentrations of Cerebrospinal fluid specimens were collected from the CA and CM patients in accordance with standard aseptic procedure and subjected to mNGS within 24 h. Next, CSF samples were subjected to bead beating, DNA extraction, DNA library construction, and sequencing . Nucleic acids extracted from blood of healthy volunteers were mixed with sterile water as negative controls. CSF specimens from three non-infected patients were sequenced as positive controls using the MGI DNBSEQ platform (BGI-Tianjin). The procedure has been described in detail elsewhere , 2020.Aspergillus species with an SSRN ≥ 2 were considered positive. The data interpretation method has been described in detail elsewhere , genome coverage (%), and depth. lsewhere .n = 50, 250, and 500) were mapped to SDSSRNs.Continuous data were subjected to non-parametric tests. Quantitative variables are expressed as medians (ranges) and qualitative variables as percentages. Data processing was performed using SPSS software . We performed a linear regression of SSRN and the number of spores that transected the origin. Because the total number of reads obtained from mNGS varied among samples, we first standardised the SSRN to obtain the standardised species-specific read number (SDSSRN) for comparison purposes. The standardised ratio (SR) was calculated as the number of total reads × (1-adaptor ratio)/20,000,000 . Twenty A. fumigatus detection given an SDSSRN of 2, the former standard for A. fumigatus detection. We hypothesised that the probability of mapping a single PMseq read to the A. fumigatus genome is a Bernoulli process, as suggested by Ebinger et al., meaning that the read either mapped correctly or incorrectly  and the number of trials . For a PMseq read, a DNA sequence of 150 bp on average is produced, and 50 bp at one end is used for genome mapping. Hrant et al. suggested that a 50 bp read obtained by shotgun-sequencing gave a 6% probability of mapping to multiple genomes and 0.52% probability of erroneous mapping  was 0.85. Therefore, we defined the detection probability as [1 – (0.15)SDSSRN], where 0.15 is the likelihood that one SDSSRN read was mapped incorrectly.We assessed the probability of orrectly . Therefo mapping , while HOf the seven non-HIV-infected patients identified, three (42.86%) were males. The median age at presentation was 46 (27–80) years. The patients’ characteristics are listed in All of the patients with CA  underwent cranial magnetic resonance imaging (MRI) and exhibited space-occupying lesions, including in the paranasal sinuses, cavernous sinus, or skull base in five cases  and frontal lobe and insular lobe in two cases  . LeptomeD-glucan (BDG) testing, and all were positive . Five patients underwent serum galactomannan (GM) testing, and three  were positive (20) was found in six patients . The CSF white blood cell count ranged from 0 × 106 to 530 × 106/L (median = 18 × 106/L), the CSF glucose level from 2.4 to 5 mmol/L (median = 3.2 mmol/L), and the CSF protein level from 0.2 to 1.238 g/L (median = 0.878 g/L). All fungal cultures of the CSF were negative , and fungal culture of the brain tissue of case 7 was positive for A. fumigatus. Twelve of the 13 cases of CM are described in detail elsewhere -β-positive . The CSFlsewhere . The sevs hyphae . PeriodiA. flavus (SSRN 2–8) in three cases. Species-specific reads mapped onto A. sydowii (SSRN 11–92) and A. oryzae (SSRN 21–23) in two cases. Among the six mNGS-positive cases, four  had more than two Aspergillus species. In the six cases with CA, the percentage of SSRNs of Aspergillus species  was 0.90% (7/774), 31.82% (7/22), 90.91% (30/33), 19.35% (18/93), 73.64% (95/129), and 43.75% (28/64), respectively. Of the 13 patients with CM, Aspergillus was found in the CSF of two  had SDSSRNs of 7,754, 11,157, and 27,253 for 50, 250, and 500 added spores, respectively. For case 2-P, the corresponding SDSSRNs were 847, 2,794, and 8,403; and for case 3-P, they were 200, 1,421, and 3,874 . As no SSRN of 2 . TherefoAspergillus species. Moreover, although Aspergillus may be present at low abundance, it should not be regarded as background contamination because of the high mortality rate.Most studies of CA are case reports or case series . In thisAspergillus contains 446 species  can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by the Ethics Committee of the Chinese PLA General Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.J-TZ designed the study. S-YY organised the experts’ meeting. R-ST, Y-BM, R-FW, G-EY, and FC contributed to the acquisition of clinical data. XT and Q-PG conducted the pathological analysis. S-FY performed the mNGS analysis of positive controls. X-WX conducted analyses and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study examined the usefulness of cerebrospinal fluid (CSF) neuron-specific enolase (NSE) levels as a candidate biomarker of neurodegeneration in Alzheimer’s disease (AD), Parkinson’s disease (PD), PD with dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).We performed a systematic search of PubMed, the Cochrane Library, Scopus, and Google Scholar to find studies that measured CSF NSE levels in AD, PD, DLB, and/or MSA. For each disease, we pooled all available data and performed a meta-analysis, and meta-regression analyses of age and sex were conducted if the main analysis found a significant association.g = 0.822, 95% confidence interval [95% CI] 0.332 to 1.311, p = 0.0010), but the data exhibited high heterogeneity . The meta-regression analysis of AD showed that age (p < 0.001), but not sex, had a significant effect on CSF NSE levels. A meta-analysis of the pooled data for PD/PDD/DLB did not show any significant changes in the CSF NSE level, but a sub-group analysis of PDD/DLB revealed significantly elevated CSF NSE levels . No significant changes in CSF NSE levels were detected in MSA.Twenty studies were included . Significantly elevated CSF NSE levels were detected in AD (Hedges’ The CSF NSE level may be a useful biomarker of neurodegeneration in AD and PDD/DLB. Age was found to affect the CSF NSE levels of AD patients.The online version contains supplementary material available at 10.1186/s13195-021-00907-3. Neuron-specific enolase  is a 78-kDa enzyme (phosphopyruvate hydratase), which is involved in glycolysis and is abundantly and ubiquitously expressed in neurons and neuroendocrinal cells, and 98% of NSE in cerebrospinal fluid (CSF) originates from the central nervous system , 2. TherThe CSF level of NSE has also been studied in neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy body disease (LBD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, these previous studies reported inconclusive or even conflicting results.Herein, we report the first meta-analysis of the CSF levels of NSE in AD, PD, DLB, and MSA.We adopted the PRISMA 2009 system for the meta-analysis. We performed a search of PubMed, the Cochrane Library, Google Scholar, and Scopus for articles published on or before September 22, 2020. The keywords used for the search were as follows: “neuron-specific enolase” AND “cerebrospinal fluid” AND (“Alzheimer” OR “Parkinson” OR “Lewy” OR “multiple system atrophy”). Non-human studies, irrelevant studies, non-English articles, and review articles were excluded. All available articles were retrieved, and the mean and standard deviation (SD) values they reported were pooled. If an article reported median, quartile, or standard error values, the data were converted to mean and SD values using a previously reported method , 8.95% CI).Effect sizes (ES) were generated based on the sample size, mean CSF NSE level, and the associated SD values. Detailed data from our previous study  are alsoWe combined the data for PD, PDD, LBD, and DLB and analyzed it under the heading “PD/PDD/DLB” because these conditions share common mechanisms, i.e., they are all synucleinopathies involving Lewy bodies , and we Q test, and the Higgins I2 index was used to measure heterogeneity across studies. I2 indexes of 0–25%, 26–50%, 51–75%, and 76–100% were regarded to indicate low, medium, high, and very high levels of heterogeneity, respectively. Forest plots and funnel plots were created with the abovementioned software. Egger’s test was used to check for publication bias. Sensitivity analyses were performed by removing the data for one study at a time to test whether the outcomes of the meta-analysis were significantly influenced by a single study. When significantly altered CSF NSE levels were detected in the main analysis, meta-regression analysis was performed to assess the effects of age and sex.All statistical analyses were performed using the STATA software, version 16 , and a random effects model (the DerSimonian-Laird method) was adopted. Heterogeneity was assessed with Cochran’s p-values of <0.05 (p < 0.05) were considered statistically significant. We did not adjust the level of significance for multiple comparisons because of the exploratory aims of our analyses.We also performed a second search of PubMed, the Cochrane Library, Google Scholar, and Scopus for articles containing information about CSF NSE levels in frontotemporal dementia (FTD) published on or before August 21, 2021. The keywords used for the search were as follows: “neuron-specific enolase” AND “cerebrospinal fluid” AND .We found 41 relevant articles in PubMed, 21 in the Cochrane Library, 127 in Scopus, and 576 in Google Scholar. We scrutinized their titles, abstracts, and contents and eliminated the articles that met the exclusion criteria. Finally, 20 studies  were included –28 , but the data exhibited very high heterogeneity , i.e., no publication bias was observed.The sensitivity analysis, which involved the removal of the data for each study in turn, did not identify any study that significantly affected the result, and therefore, the consistency of the conclusion was confirmed. A funnel plot is shown in Fig. 95% CI 0.0828 to 0.2424, z = 3.99, p < 0.001), but not sex (p > 0.05), had a significant effect on the CSF NSE levels of AD patients.The meta-regression analysis showed that age , although the data exhibited very high heterogeneity . However, a sub-group analysis showed significantly elevated CSF NSE levels in PDD/DLB , i.e., there was no publication bias.The pooled data for these diseases are summarized in Additional file g = 0.494, 95% CI 0.179 to 0.810). In a meta-regression analysis of PDD/DLB, neither age nor sex exhibited a significant effect on the CSF NSE level (p > 0.05).In the sensitivity analysis, the results were only affected when the data from the study by Abdo et al.  were remWhen the subjects were pooled, the mean age of the PD/PDD/DLB patients was 64.0 ± 17.7 years , and that of the controls was 61.1 ± 16.5 years.The pooled data for MSA are summarized in Additional file Two studies examined MSA with predominant Parkinsonism (MSA-P) and MSA with cerebellar features (MSA-C) separately , 28. We g = 0.387, 95% CI −0.293 to 1.067, p = 0.2648; Fig. Q = 20.63, df = 3, p = 0.0001, I2 = 85.46%). Furthermore, sub-group analyses of MSA-C and MSA-P without the data from the study by Santaella et al. [g = 0.412, 95% CI −0.654 to 1.479; MSA-P: Hedges’ g = −0.006, 95% CI −0.577 to 0.566; see Additional file Meta-analysis did not show any significant changes in CSF NSE levels in MSA , which suggested that our data for MSA were affected by publication bias.A funnel plot is shown in Fig. When the subjects were pooled, the mean age of the MSA patients was 62.1 ± 8.2 years, and that of the controls was 58.9 ± 11.9 years.This is the first reported meta-analysis of the CSF NSE levels of AD, PD, DLB, and MSA patients, and it provided evidence about the significance of CSF NSE levels in AD and PDD/DLB.This study detected significantly elevated CSF NSE levels in AD patients, which are considered to reflect the neurodegenerative processes that occur in AD, and the sensitivity analysis of the AD-related data confirmed the consistency of the results. Therefore, this study indicated that the CSF NSE level might be useful as an objective surrogate biochemical marker of AD-related neuronal damage. Total tau (t-tau) is widely accepted as a biomarker of AD-related neurodegeneration , and theMeta-regression analysis revealed that age contributed to the high heterogeneity of the CSF NSE data, and this point should be considered when interpreting the CSF NSE levels of AD patients. However, a previous study reported that CSF NSE levels were not correlated with age or sex in a normal population . Other pThe high heterogeneity observed in the CSF NSE levels of the AD patients in this study raises other possibilities, such as the effects of diagnostic accuracy. The clinical diagnosis of AD is based on the relevant criteria, but post-mortem pathological verification has demonstrated the difficulty of achieving an accurate pre-mortem diagnosis and differentiating AD from non-Alzheimer’s dementias, such as argyrophilic grain disease, primary age-related tauopathy, and other AD-mimicking disorders, pre-mortem . A previOur study could not elucidate the effects of other confounding factors, such as disease severity, because relevant information was missing from the source data. For example, we checked the Mini-Mental State Examination (MMSE) scores of the subjects in previous studies , Fms-related tyrosine kinase ligand (Flt3L), YKL-40 , and ubiquitin carboxy-terminal hydrolase L1 (UCHL-1) . FurtherWe focused on neurodegenerative disorders related to AD pathology (amyloid β and tau) and synucleinopathies in the initial stage of this study. We subsequently tried to perform a second search for studies relating to FTD; however, the number of studies found was too small to allow a meta-analysis to be performed. It should also be mentioned that the clinical, pathological, and genetic features of FTD exhibit marked heterogeneity . FurtherThe assays used in previous studies included enzyme-linked immunosorbent assays, electrochemiluminescence immunoassays, and radioimmunoassays. The validity of direct comparisons among these methods has not been fully elucidated, but each method is well established, and therefore, data obtained using these methods should be comparable. Some previous studies mentioned coefficients of variation (CV) for intra- or inter-assay verification and dynamic ranges for CSF NSE measurements, and other studies used commercial kits, for which CV and dynamic ranges can be obtained from the manufacturer’s information. Further studies are needed to standardize CSF NSE measurement methods.This study had some limitations. First, the CSF NSE level data exhibited high heterogeneity, and there were quite large overlaps between the disease groups and controls. Therefore, the application of this biomarker to clinical practice should be performed cautiously. Second, elevated CSF NSE levels reflect neuronal damage, but are not disease-specific. Several of the studies included in this study used panels of biomarkers  to detect combinations of molecular pathological changes.Nevertheless, measuring CSF NSE levels could be useful because NSE assays are available in many laboratories.This meta-analysis revealed significantly elevated CSF NSE levels in AD and PDD/DLB, but not in PD without dementia or MSA. This study will aid our understanding of the pathological mechanisms underlying these diseases and support further investigations, more accurate diagnosis, and evaluations of therapeutic interventions.Additional file 1.. Pooled data for Alzheimer’s disease. SD: standard deviation; SE: standard error.Additional file 2.. Pooled data for Parkinson’s disease (PD), PD with dementia, and dementia with Lewy bodies.Additional file 3.. Pooled data for multiple system atrophy.Additional file 4.. Pooled data for frontotemporal dementia.Additional file 5.. Forest plot for the sub-group analyses of multiple system atrophy (MSA). CI: confidence interval; MSA-C: MSA with cerebellar features; MSA-P: MSA with predominant Parkinsonism; SD: standard deviation.