Preeclampsia (PE) is an important topic in obstetrics. In this study, we used weighted gene co-expression network analysis (WGCNA) to screen the key modules related to immune cell infiltration and to identify the hub genes for the molecular subtyping of PE. We first downloaded a set of PE transcriptional data  from the GEO database. We then analyzed the PE samples and non-PE samples for immune cell infiltration and screened cells with differences in such infiltration. Next, we downloaded the immune-related genes from an immune-related database to screen the expression profile of the immune-related genes. Then, we obtained a candidate gene set by screening the immune-related genes differentially expressed between the two groups. We used WGCNA to construct a weighted co-expression network for these candidate genes, mined co-expression modules, and then calculated the correlation between each module and immune cells with differential infiltration. We screened the modules related to infiltrating immune cells, identified the key modules’ hub genes, and determined the key module genes that interacted with each other. Finally, we obtained the hub genes related to the infiltrating immune cells. We classified the preeclampsia patients by unsupervised cluster molecular typing, determined the difference of immune cell infiltration among the different PE subtypes, and calculated the expression of hub genes in these different subtypes. In conclusion, we found 41 hub genes that may be closely related to the molecular typing of PE.  Preeclampsia (PE) is a pregnancy-specific hypertensive disease, in which individuals with normal blood pressure before pregnancy develop hypertension, urinary protein, and other features after 20 weeks of pregnancy . PE affeAlthough the exact cause of PE remains unclear, its clinical manifestations are thought to be the result of endometrial dysfunction caused by placental dysfunction, characterized by inadequate uterine spiral artery transformation. This placental defect is most likely associated with partial breakdown of the mother’s immune tolerance . At the Many studies using bioinformatics for screening to identify PE-related genes have been performed. Many important genes and pathways related to PE, including TLRs , the MAPThe aim of this study was to screen the key modules related to PE immune infiltration based on WGCNA and identify the hub genes for PE molecular subtyping. The differential expression of hub genes among different PE subtypes suggests that these genes are potential markers for PE subtyping.https://www.ncbi.nlm.nih.gov/gds), as shown in First, we downloaded a set of PE chip data, GSE75010, from the GEO database (https://www.immport.org/), TITIIDB  of the probability of occurrence of the node, and the correlation coefficient was greater than 0.8, which was consistent with the scale-free network of the co-expression network. To ensure that the network was scale-free, we selected the optimal soft threshold of 5. In the next step, we transformed the expression matrix into an adjacency matrix, which we then converted into a topological matrix. Based on topological overlap matrix (TOM) , we clusThen, we calculated Pearson’s correlation between the five modules and the clinical traits of differentially infiltrated immune cells. We found that the blue and brown modules were most associated with immune cell infiltration. We then calculated Pearson’s correlation coefficient in the most relevant blue and brown modules between immune genes and the modules and clinical features, respectively.First, we selected the genes with a gene significance coefficient greater than 0.5 and a module membership coefficient greater than 0.5 as candidate hub genes. Then, we constructed a PPI network of the blue and brown modules and selected a connectivity threshold of 0.1, which led to the recognition of a total of 120 nodes. Finally, we obtained 41 critical hub genes that overlapped between the 49 hub genes and 120 nodes.We also performed disease classification of the PE samples by using the identified hub genes and determined the optimal K value (number of classes) by searching for the best SSE. We classified PE into different subclasses by unsupervised clustering K-means combined with TSEN dimension reduction. We examined the expression patterns of these hub genes in the different subclasses and analyzed the significantly differentially expressed genes in the different subclasses . These differentially expressed genes are potential marker genes of PE subclasses.All of the analyses were performed using R (V. 4.0.2) statistical software. The differentially expressed genes were calculated using the R package limma. The enriched functions associated with the differentially expressed genes were determined using the R package clusterProfiler . The co-In this study, we analyzed microarray datasets from the GEO database. We used Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) to analyze differentially infiltrating immune cells in PE. We also analyzed the biological processes and signal transduction pathways related to the DEGs, and evaluated the ability of gene signatures to predict the PE subclass. Here, we screened 41 critical hub genes by comparing the immune cell infiltration and classified the samples into five subclasses according to the hub genes. We found that most of the genes were expressed at a low level in cluster 1 and a high level in cluster 2, suggesting that these genes are potential markers in different PE subtypes.Using the CIBERSORT LM22.txt file as the feature gene set, we analyzed the immune cell infiltration in PE and normal samples. The results showed that the infiltration of PE samples was significantly different from that of normal samples (p < 0.05) regarding B cells naive, plasma cells, T cells CD8, T cells regulatory (Tregs), Macrophages M0, Macrophages M2, mast cells resting, eosinophils, and neutrophils .Figure Setting p < 0.05 as a threshold, we obtained 428 differentially expressed immune-related genes, of which 181 were upregulated in PE and 247 were downregulated .Figure For the 428 differentially expressed immune-related genes, we performed GO and KEGG enrichment analyses, and found that these genes were particularly associated with immune-related functions, such as inflammatory response, cytokine production, cytokine–cytokine receptor interaction, and biological functions related to the MAPK immune signaling pathway .Figure We used the R package WGCNA software to construct a weighted co-expression network of candidate differentially expressed immune-related gene sets. We found that the log(k) of the node with degree K had a negative correlation with the log[p (k)] of the probability of occurrence of the node, and the correlation coefficient was greater than 0.85, which is consistent with the scale-free network of the co-expression network. To ensure that the network was scale-free, we selected the optimal soft threshold of 5 .Figure The next step was to transform the expression matrix into an adjacency matrix, which we then converted into a topological matrix. Based on TOM, we used the average-linkage hierarchical clustering method to cluster genes according to the criteria of the hybrid dynamic shear tree and set each gene network module to at least 30 genes. After determining gene modules by the dynamic shear method, we calculated the eigengenes of each module at one time, and then clustered the modules, merged the nearby modules into new modules, and set height equal to 0.25. This led to five modules being obtained . We counWe selected the differentially infiltrating immune cells shown in http://www.cytoscape.org/) based on blue and brown modules . We selected the genes with a gene significance coefficient greater than 0.5 and a module membership coefficient greater than 0.5 as the key genes to identify 49 potential hub genes, of which 40 were blue and 9 were brown , B. We tFrom the clinical features downloaded from GEO, we analyzed the correlation of these 41 hub genes with the clinical features of advanced-age pregnancy, maternal hyperproteinuria, maternal hypertension, maternal abortion, HELLP syndrome, and intrauterine growth retardation (IUGR). The results showed that LANCL1 and PROS1 may be associated with advanced-age pregnancy; LST1 and VAV1 may be associated with maternal hyperproteinuria; BMP5 may be associated with maternal history of hypertension; FCER1G, IFI16, MKNK1, and Pik3cg may be associated with maternal history of abortion; bMP5, Cysltr1, FGF7, IRAK1BP1, and LST1 may be associated with HELLP syndrome ; and c3AR1, CD200R1, CD28, CYBB, FCER1G, FGF10, IFI16, IL12RB2, Kitlg, LGR5, NCKAP1L, PIK3CG, Rarb, TLR7, and VAV1 may be associated with IUGR .Figure Based on the analysis results of the WGCNA co-expression modules, we further analyzed 41 hub genes identified by blue and brown modules and subjected them to unsupervised clustering. We selected PE samples (N = 80) to extract the expression profile data of these 41 hub genes. We classified all PE samples by the K-means unsupervised clustering method. First, we selected the optimal K value by searching for the inflexion of the sum of squares due to error . As shown in Using LM22.txt obtained from the official CIBERSORT website, we analyzed cluster 1 and cluster 2 for immune cell infiltration. The results in To explore the expression of these genes in the different PE subclasses, we calculated the expression of the hub genes in the different preeclampsia subtypes based on As a hypertensive disorder complicating pregnancy, PE is one of the leading causes of maternal mortality globally. PE-induced fetal growth restriction, preterm birth, and other common complications also place huge economic and psychological costs on the affected family and society at large. Maternal immune tolerance, which plays an important role in the pathogenesis of PE, and the remodeling of spiral arteries are closely related to the infiltration of placental immune cells. Immune cell infiltration is a novel bioinformatic approach that has been used to investigate the diagnosis and prognosis of a variety of diseases including colon , gastricHere, we obtained the gene expression profiles of 7,859 genes by filtering the gene chip data downloaded from GEO. We obtained 2496 immune-related genes from the immune-related database and 922 genes with expression profile data. We screened a total of 428 differentially expressed immune-related genes  using LIMMA. We used the R package clusterProfiler for enrichment analysis of these differentially expressed genes and found that they were linked to immune-related functions such as inflammatory response, cytokine production, cytokine–cytokine receptor interaction, and immune signaling pathway-related biological functions . This is consistent with previous studies that reported that the above biological processes and pathways play important roles in the development of PE. Using proteomic techniques, it has been shown that proteins related to lipid metabolism and inflammatory pathways can be used as early diagnostic markers of PE . In addiUsing WGCNA to construct a weighted co-expression network of these genes, four co-expression modules were obtained, among which the immune-related genes of the blue and brown modules were those most strongly related to the infiltrating immune cells; the immune-related genes of the blue module numbered 120, while those of the brown module numbered 62. Further analysis of the genes of the most relevant blue and brown modules and the genes of the PPI interaction network that was constructed yielded 41 hub genes. These hub genes included LANCL1 and PROS1, which may be associated with advanced-age pregnancy; LST1 and VAV1, which may be associated with maternal hyperproteinuria; BMP5, which may be associated with a maternal history of hypertension; FCER1G, IFI16, MKNK1, and Pik3cg, which may be associated with a maternal history of abortion; BMP5, CYSLTR1, FGF7, IRAK1BP1, and LST1, which may be associated with HELLP syndrome ; and C3AR1, CD200R1, CD28, CYBB, FCER1G, FGF10, IFI16, IL12RB2, KITLG, LGR5, NCKAP1L, PIK3CG, RARB, TLR7, and VAV1, which may be related to intrauterine growth restriction (IUGR).Among these, BMP5 , CD200R1In addition, immune cell infiltration was analyzed in preeclampsia and normal samples by CIBERSORT. Nine immune cells, namely, B cells naive, plasma cells, T cells CD8, T cells regulatory cells (Tregs), macrophages M0, macrophages M2, mast cells resting, eosinophils, and neutrophils, were screened. We found significantly different infiltration between cluster 1 and cluster 2 of four types of immune cell: dendritic resting cells, macrophages M2, NK cells resting cells, and NK cells activated cells. The literature shows that decidual NK cells promote trophoblast invasion by secreting chemokines, and decidual macrophages act as antigen-presenting phagocytes, secreting cytokines, and regulating the immune balance between mother and fetus . T cellsAccording to the expression of the 41 identified hub genes in patients with PE in this study, we classified the samples by the K-means unsupervised clustering method, leading to the samples being grouped into five categories. We found that most of the genes were expressed at a low level in cluster 1 and a high level in cluster 2, suggesting that these genes are potential markers in different subtypes of preeclampsia. This parallels a study by Liu et al., in which EGR1, LEP, and HBB were used as DEGs to identify early-onset and late-onset PE . In addiThis study also has some shortcomings. First, although PE was divided into five clusters according to the hub genes, the numbers of samples in clusters 3, 4, and 5 were too small to be used for infiltration analysis. In future work, the sample size should be expanded and a more complete database should be established, enabling further analysis of the other clusters. Second, although 41 hub genes were identified as potential biomarkers for PE immunotyping, no in vivo or in vitro studies were carried out, so this should be a focus in future work.Using WGCNA, we divided the genes specifically differentially expressed in PE into four modules. We selected the two modules with the highest correlation with immune cell infiltration for a PPI network and obtained 41 hub genes. We classified PE samples into five clusters according to the unsupervised clustering of hub genes and found that cluster 1 and cluster 2 had significant differences in immune cell infiltration and hub gene expression. This work suggested that hub genes can be used to classify PE into different subtypes.Click here for additional data file.


To evaluate the value of non-invasive detection of advanced glycation end products (AGEs) in the early screening of type 2 diabetes mellitus (T2DM) in the community of China.From January 2018 to January 2019, a total of 912 patients with community health physical examination and no history of T2DM were selected, excluding the results of missing value > 5%. Finally, 906 samples were included in the study, with a response rate of 99.3%. Non-invasive diabetic detection technology was used to detect AGEs in the upper arm skin of all participants, AGE accumulations were classified as ≤P25, P25∼P50, P50∼P75, and >P75; HbA1c, insulin, C-peptide, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), creatinine, urea, and other indicators were measured at the same time. Univariate analysis of variance was used to compare the differences in general data, biochemical indexes, skin AGE levels, and blood glucose among groups, and logistic regression analysis and latent category analysis were performed.In univariate analysis, SBP, FBG, HbA1c, and age were correlated with higher AGE (p < 0.01); TG, TC, HDL, UA, and gender were not positively correlated with AGE (p < 0.01). After controlling for covariates , AGE accumulation was interacted with other variables. The results of latent category analysis (LCA) showed that the health risk factors (HRFs), including age, systolic blood pressure, HbA1c, FBG, triglyceride, total cholesterol, HDL-C, and uric acid, were divided as three groups, and AGE is divided into four categories according to the quartile method, which were low risk (≤P25), low to medium risk (P25∼P50), medium to high (P50∼P75), and high risk (>P75), respectively. The association between the quartile AGE and risk factors of the OR values was 1.09 , 2.61 , and 5.41 , respectively. The moderation analysis using the PROCESS program was used to analyze whether BMI moderated the link between risk factors and AGE accumulation. There was also a significant three-way interaction among HRFs, BMI, and gender for AGE accumulation in the total sample (β = -0.30).Non-invasive skin detection of AGEs has a certain application value for the assessment of T2DM risk and is related to a variety of risk factors. Therefores/en/) . The exires/en/) . Previoures/en/) . Many rires/en/) , 8. Moreres/en/) . AGEs bires/en/) . Zhu et res/en/) . In addires/en/) . Besidesres/en/) , furtherAt present, clinical management of diabetes mainly focuses on blood glucose control, blood pressure control, lipid lowering, lifestyle, and other risk factors. If blood glucose is a major factor in the occurrence of these complications, then by normalizing blood glucose levels , diabeteAGEs can cause islet mitochondrial dysfunction, resulting in excessive superoxide production and reduced ATP level, and eventually lead to insulin secretion dysfunction, leaving the body in a state of continuous hyperglycemia. After AGEs and RAGE (receptors of AGE) were combined, the expression of the Bcl-2 gene in islet β cells was decreased, leading to mitochondrial swelling and apoptosis, and impaired glucose aerobic oxidation, which further enhanced the formation of AGEs. In addition, AGEs can also induce pancreatic beta cell apoptosis by inhibiting the autophagy of beta cells, and AGEs can directly damage islet β cells, which is related to the occurrence and development of type 2 diabetes mellitus (T2DM). Moreover, AGEs and their receptors may play important roles in the development and progression of coronary artery disease (CAD) in patients with T2DM . MoreoveAt present, a large-sample invasive serological examination is difficult to be popularized in some grassroots and communities, and there is still a lack of relevant reports on whether non-invasive skin detection of AGEs is suitable for early risk screening of T2DM. Therefore, the author aims to explore the application value of the non-invasive T2DM detector in the early screening of large-scale T2DM in the Chinese population by extensively carrying out the non-invasive and rapid skin detection project of AGEs in the community.Using the multistage sampling method, from January 2018 to January 2019, three community health service centers in Hefei that signed the medical consortium agreement with the First Affiliated Hospital of Anhui Medical University were selected to screen the population aged 30 to 80 years in each community. The exclusion criteria are as follows: previous history of T2DM, severe cardiovascular and cerebrovascular diseases, liver and kidney function impairment, infection or stress, and people in the terminal stage of various diseases.Doctors and personnel with professional training in designated physical examination institutions had undergone physical checkups based on standard procedures, such as height, weight, AGE test, fundus examination, blood pressure, and blood and urine samples.Gender, age, height, weight, waist circumference, hip circumference, blood pressure, and other information of patients in all participants were recorded, and the body mass index (BMI) and waist–hip ratio (WHR) were calculated. Fasting venous blood was collected, and measured fast plasma glucose (FPG), 2h plasma glucose (2hPG), glycated hemoglobin A1c (HbA1c), insulin, and C-peptide were tested by the oral glucose tolerance test (OGTT); total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), creatinine, urea, and other indicators were tested by blood serum. These risk factors are collectively referred to as health risk factors (HRFs). At the same time, the non-invasive T2DM detector  was used to detect the fluorescence of the subjects’ upper arm skin. Obvious blood vessels, scars, and malformed skin areas should be avoided during measurement. Selected test sites should be wiped with alcohol to obtain the fluorescence spectrum intensity of AGEs on subjects’ skin (unit: AU). Measurements should be made for 3 times, and the average value should be taken as the final skin AGE testing results. The ethics committee approval number is PJ2019-09-05.via systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg (WHO 1999 criteria) (via serum concentrations of TC>5.69 mmol/l or TG>1.68 mmol/l or HDL>1.03 mmol/l or patients that have been treated with medications for lowering of lipids. Overweight was defined as a BMI >24 and <28 kg/m2. Obesity was characterized as BMI ≥ 28 kg/m2 based on Chinese standards (via serum uric acid level ≥ 416 mmol/l (≥7.0 mg/dl) in men and ≥386 mmol/l (≥6.5 mg/dl) in women or if they were treated with allopurinol for lowering the level of uric acid (Hypertension was characterized riteria)  or curretandards . Hyperurric acid . The ageric acid . FBG  was used to derive distinct variable centered HRFs clusters, namely groups of HRFs that tend to co-occur across participants. This approach helped us to reduce the risk of overfitting. In the analysis, each FA-derived cluster was indexed by a binary variable indicating the presence or absence of any exposure to the relevant HRFs throughout childhood. Third, latent class analysis (LCA) was performed using Mplus in order to derive HRFs clusters.All the data were analyzed using SPSS 23.0. Statistical analyses were carried out in three main steps. Missing data on the outcome and exposure variables were estimated using multiple imputations by SPSS 23.0. Latent class analysis (LCA) was used to identify homogeneous, mutually exclusive “patterns” of 6 HRFs using MPlus 7.4. Model building begins with class enumeration, and with the addition of each subsequent latent class, goodness-of-fit indices are compared. The HRF classes were adopted based on model fit indices: Akaike information criteria (AIC), Bayesian information criteria (BIC), and sample size-adjusted BIC (aBIC), entropy, and p-value for the Lo–Mendell–Rubin test (LMRT).The specific statistical analyses were carried out in four main steps. Step 1 entailed the descriptive statistics associated with the health risk factor by AGE accumulation. In Step 2, multilevel logistic regression was used to test the association between risk factors and AGEs. In Step 3, according to the principle of LCA, the Mplus program was used to perform LCA. In Step 4, the PROCESS program of moderation was used to perform a moderation analysis . For tesχ2 = 25.76), FBG (χ2 = 37.38), SBP (χ2 = 8.46), and older age (χ2 = 231.11) were more likely to have a higher prevalence of AGE accumulation; others such as HDL (χ2 = 4.29), UA (χ2 = 2.71), TC (χ2 = 6.24), TG (χ2 = 3.90), and gender (χ2 = 1.94) have no correlation with AGE accumulation.The demographic data of all participants , physical examination , T2DM number and type of glycemic index , renal function, blood urea nitrogen, serum creatinine, blood uric acid, and blood lipid ) are shown in p < 0.01). All remaining results are reported specific to the three-class solution.As shown in In the model adjusted for gender, waist circumference, and hip circumference, children exposed to 3+ HRFs had significantly higher AGE levels than those who did not have any HRF . These datasets are shown in The statistical results of the EFA of HRFs in the training dataset are shown in In Moderation analyses were performed with waist circumference and hip circumference as the control variables. The results are presented in As shown in The earlier discussion evaluates the value of noninvasive detection method to prevent the development of the disease and shows the effectiveness of targeted prevention, including through the establishment of variables based on the genetic risk of equations to evaluate the risk of diabetes and prediabetes, and guide therapy, which reduces blood pressure, body weight, and cholesterol . AGEs acWith the development of economy, the prevalence rate of type 2 diabetes is increasing year by year, this disease should be brought into the scope of policy for key prevention and control of chronic diseases, and in the hope that prevention and control mark the service in basic hospitals, such as level 3 first-class hospitals, and incorporate the same in the community hospitals. However, because of T2DM knowledge is not enough, limited community hospital and patient factors such as fear traumatic examination, blood glucose monitoring still cannot spread, leading to low diagnostic rate and population. Therefore, it is necessary to strengthen basic diabetes screening, such as non-invasive AGE testing. In recent years, a large number of studies have confirmed that AGE is one of the important mechanisms for the occurrence and development of chronic complications of T2DM. Studies have found that excessive AGE is one of the important mechanisms leading to type 2 T2DM and its chronic complications . The accOur analysis revealed several key findings. People at potential risk of disease exposed to three or more HRFs had higher AGE accumulation, which further risks toward T2DM and its complication. Among different types and clusters of HRFs, HbAIc and FBG had the largest associations with AGE accumulation. BMI was also associated with higher AGE accumulation among participants exposed to multiple HRFs. Further, the moderation analysis indicated that gender, BMI, and HRFs were associated with AGE accumulation exposure. The relationships among HRFs, BMI, and AGE accumulation generally had an interaction effect.2) exhibited significantly higher-circulating AGEs compared to overweight participants (average BMI 26.3 kg/m2)  . Serum A3 kg/m2) . Another3 kg/m2) . Similar3 kg/m2) .Our research suggests that participants, especially those with a higher BMI and higher health risk factors, seem to be a vulnerable group for AGE accumulation. From a developmental disease perspective, obesity interacts with health risk factors, influencing the development of AGE accumulation. BMI, as a moderating variable, has gender-specific effects on the association between HRFs and AGE accumulation. The quality of gender moderates the relationship between BMI and AGE accumulation. As we all know, HbA1c and obesity were all associated with AGE accumulation; AGEs may play a more prominent role in the health of obese individuals, so obesity may be one of the health risk factors and pathophysiology mechanisms leading to AGE accumulation in health risk factors , 41. MorFluorescent intensity of AGEs in the skin can reflect the longer time of blood glucose control, which may be one of the indicators to predict the chronic complications and mortality of T2DM . PreviouThe following are the study’s limitation: a large number of foreign studies have reported that skin autofluorescence (SAF) can be used for T2DM screening, and the sensitivity of the diagnosis of T2DM is higher than FPG or HbA1c. The authors did not investigate the subjects’ family history of T2DM, dietary habits, etc., which may affect the results of the study . In addiThis study was based on existing literature documenting possible associations between AGE accumulation, BMI, and health risk factors in a Chinese cultural context. Health risk factors should be taken into account when designing interventions for AGE-cumulative primary prevention in participants, especially in women. Reducing the incidence of health risk factors can significantly improve BMI at lower levels. In addition, strengthening a lower BMI can prevent or reduce AGE accumulation, especially in women. The study also suggests that Chinese participants can use weight control therapy as an adjunct to adjusting BMI when treating adolescents with higher HRFs. In addition, with BMI as a moderating variable, the relationship between HRF and AGE accumulation is different between sexes. The findings point to the necessity to focus on normal BMI and use gender-specific approaches to address AGE accumulation in patients with a history of higher HRFs.In general, non-invasive skin detection of AGEs has a certain application value for the assessment of T2DM risk but may not be fully applicable for early screening of T2DM. Much has yet to be done in AGE research. More studies are needed to confirm this non-invasive technique as a new T2DM screening tool to improve the diagnosis rate of T2DM.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Anhui Medical University Ethics Committee . The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.QZ constructed the study design. FD and HH recruited the participants. YZ, FD, and TJ were involved in the statistical analysis. QZ was responsible for the critical revision of the manuscript. YZ, TJ, and CL edited and revised the manuscript. YZ and CL prepared and drafted the manuscript. All the authors who contributed to the manuscript gave their approval for its submission. The work presented here has not been published previously and is not being considered for publication elsewhere. All authors contributed to the article and approved the submitted version.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: funding for the project was provided by National Natural Science Foundation of China (81970703).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.


Annona muricata called acetogenins have important biological activities, such as anticancer, antiparasitic, and insecticide. Previous studies propose an inhibitory activity of bovine mitochondrial respiratory complex I by bis-tetrahydrofurans acetogenins such as annocatacin B, however, there are few studies on its inhibitory effect on human mitochondrial respiratory complex I. In this work, we evaluate the in silico molecular and energetic affinity of the annocatacin B molecule with the human ND1 subunit in order to elucidate its potential capacity to be a good inhibitor of this subunit. For this purpose, quantum mechanical optimizations, molecular dynamics simulations and the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) analysis were performed. As a control to compare our outcomes, the molecule rotenone, which is a known mitochondrial respiratory complex I inhibitor, was chosen. Our results show that annocatacin B has a greater affinity for the ND1 structure, its size and folding were probably the main characteristics that contributed to stabilize the molecular complex. Furthermore, the MM/PBSA calculations showed a 35% stronger binding free energy compared to the rotenone complex. Detailed analysis of the binding free energy shows that the aliphatic chains of annocatacin B play a key role in molecular coupling by distributing favorable interactions throughout the major part of the ND1 structure. These results are consistent with experimental studies that mention that acetogenins may be good inhibitors of the mitochondrial respiratory complex I.ND1 subunit possesses the majority of the inhibitor binding domain of the human mitochondrial respiratory complex I. This is an attractive target for the search for new inhibitors that seek mitochondrial dysfunction. It is known, from in vitro experiments, that some metabolites from  It has been almost 100 years since Warburg presented the first connection between the mitochondria and tumors appearance ,3. On thFor instance, the mitochondrial respiratory complex I (MRC-I) is directly involved in the appearance of colorectal cancer , prostatFabaceae plants. Furthermore, it was used as a pesticide and piscicide [MRC-I, also named ubiquinone oxidoreductase , has a molecular mass of approximately 1 MDa; its structural conformation is composed of fourteen central subunits. ND1 subunit is one of those and has most of the inhibitor binding domain in the ubiquinone oxidoreductase. To date, one the main known inhibitors of the MRC-I is the rotenone molecule . Rotenoniscicide  due to iiscicide ,17. Its iscicide ,19. Neveiscicide ,20,21. CAnnonaceae, i.e., soursop (Annonamuricata) [Murai et al. analyzed rotenone and a synthetic acetogenin as an inhibitor of the bovine heart MRC-I . They reuricata) .Annonaceae plants [In traditional medicine, soursop also has important uses, including anticonvulsant, antiarthritic, antiparasitic, hepatoprotective, etc. Many of these beneficial attributes have been ascribed to acetogenins ,25. One e plants ,25,27. Te plants .Acetogenins have showed important behaviors when evaluating their potential cytotoxic activity against cancer cells; some of these molecules already have proven anti-cancer properties, such as bullatacin, motrilin, assimin, trilobacin, annonacin, gianttronenin, and squamocin. However, we still do not have enough information about most of the acetogenins . The maiSCRF = ), and molecular electrostatic potential (ESP) surfaces were used to visualize the polar and non-polar regions of these ligands. To obtain the MD parameters and topologies of the ligands, we used the TPPMKOP server [We analyzed two molecules as ligands to the ND1 complex, rotenone (PubChem ID 6758) and annocatacin B (PubChem ID 10483312) a. The stP server , which uP server ,40. Thesx and y directions), to obtain the membrane model. The InflateGRO methodology was used for the embedding of ND1 protein in the lipid membrane [On the other hand, the phospholipid bilayer membrane was built with 512 dipalmit- oyl-phosphatidylcholine (DPPC) molecules. A 128-DPPC bilayer with 64 lipid molecules in each layer was replicated four times  by the PDB ID: 5XTD . Crystalz axis. SPC water-model molecules and ions were added to neutralize the systems. Next, we proceeded with another energy minimization with a maximum of 50,000 steps. The equilibrium MD simulation was realized with position restraint in two ensembles. The first was the canonical ensemble (NVT) at 323.15 K with a trajectory of 50 ps using a V-rescale thermostat. The second was the isobaric-isothermal ensemble (NPT) at 309.65 K, with semi-isotropic pressure coupling, the compressibility of 4.5 × 10Molecular dynamics (MD) simulations were carried out in Gromacs 2019  with theFirst, the coupling was made between the ND1 subunit and rotenone, and then, between ND1 and annocatacin B. To accomplish this, we used PATCHDOCK server ,47, a moTo evaluate the binding affinities of ND1-ligand interactions, we performed the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations . This waal costs ,51,52. TIn Equation , the bon as zero . The nonEquation , the sole energy . Finallygmx sham module, while the RMSD and RG were considered as the atomic position variables with respect to its average structure and figures were constructed using Wolfram Mathematica 12.1 [Statistical results, root mean squared deviation (RMSD), root mean squared fluctuation (RMSF), radii of gyration (RG), solvent accessible surface area (SASA), hydrogen bonds (HB), binding free energies (BFE), matches, structures, trajectories, B-factor maps, were obtained using Gromacs modules. An analysis of structure properties was performed using the MD trajectories of the last 200 ns of each simulations, then visualized using Visual Molecular Dynamics (VMD) software  and UCSFica 12.1 .The human MRC-I belongs to a highly organized supercomplex, named respirasome. The complexes I, III, and IV arise in a more stable fashion at that supercomplex and have the special task of channeling electrons effectively through the electron transport chain . NeverthMRC-I is the first in the mega-complex that encounters the quinone site to start the oxide-reduction process. This complex is composed of several subunits, and mainly the ND1 subunit is the one that possesses the majority of the quinone binding domain and, to a lesser extent, the ND3, PSST, and 49 kDa subunits. Fiedorczuk et al. studied the open and close positions of the above-mentioned complex I to be active and inactive, respectively . The ND1Before performing the MD simulations, we carried out QM calculations to obtain the optimized structures and analyze the electrostatic properties of the ligand molecules. one ring b. This r bilayer .Both ligand molecules have an electrophilic character and one of the major goals of this study aimed to assess the electrostatic effect of the ligands on the ND1 structure. HF rings a. On theHF rings b. With tAdditionally, the drug-like properties of annocatacin B and rotenone have the following values: six hydrogen bond acceptors in both of them; hydrogen bond donors of 2 and 0; molecular weight of 578.875 g/mol and 394.423 g/mol; the number of rotational bonds of 23 and 3; partition coefficient LogP of 8.1069 and 3.7033, and a surface area of 250.531 ÅThe pharmacokinetic properties of absorption, distribution, metabolism, excretion, and toxicity (ADMET) are in As we said earlier, ND1 subunit is located in the transmembrane region of human MRC-I . In ordeBefore studying the structural and energy changes of ND1 protein, it was necessary to assess the stability of the molecular complexes during MD simulations. For this purpose, we calculated and plotted the root mean square deviation , close values were obtained for the ND1-ligand complexes (∼2.12 nm), due to the stability provided by the lipid membrane . Similary-axis RG, we performed a free energy landscape (FEL) analysis to obtain the minimal global energy conformations of the ND1 complexes. The FEL maps showed the impact of ligands on the ND1 structure stabilization. In Using both results, the active site RMSDs and hbond tool of Gromacs for the MD simulations, and the Hydrogen bonds plugin of VMD for the global minimum energy structures, we obtained the HB interactions based on a cutoff distance of 0.35 nm and a cutoff angle of 30To elucidate this apparent contradiction between the results obtained from the RMSD and RG analyses, we performed a hydrogen bonds (HB) analysis. Using the olecular . The resTo clarify these stability behaviors, we carried out HB calculations between the ligand molecules and the system components. In To evaluate local flexibilities of the ND1 subunit and describe the deviations of residues from the average position due to the ligand effects, we performed the root mean square fluctuation (RMSF) analysis. The main fluctuations of the ND1 protein were observed at the unembedded-loop regions, as expected . In partDespite the high stability of the active site, a fluctuation analysis was performed to understand the ligand effect in these region. For this purpose, in addition to RMSF calculations, we analyzed the B-factor, also called thermal factor or Debye–Walle factor  and we mThe stable regions in the MD trajectories were used and the nearest neighbor residues B-factor values are shown in 2) than ND1—annocatacin B (177.67 ± 1.53 nm2) and ND1 without ligand (174.84 ± 1.37 nm2).Finally, we included the solvent accessible surface area (SASA) value, that is an important descriptor of the ligand effects over the structure, in which ND1—rotenone has a higher value  approximation , we calcThe electrostatic map of the ND1 structure shows a well-defined charged core surrounded by hydrophobic alpha-helices substructures. The core is formed mainly by the active site residues that confer a high electrophilic character to this region . We compared these results with those obtained through the analysis of the ND1—rotenone complex, owing to the fact that the rotenone molecule is a powerful inhibitor of this MRC-I. ND1 subunit is a transmembrane protein, thus we used a DPPC lipid bilayer as a membrane model for the entire simulations of the ND1 complexes in this work.The overall analysis revealed a stabilizing effect of the annocatacin B molecule over the ND1 protein structure. Mainly, this could be due to the size and its capability of folding of annocatacin B, which ultimately allowed it to have more interactions with the ND1 nearby residues. We observe an increase in the formation of hydrogen bonds in the ND1—annocatacin B complex through the whole MD trajectory, which suggests a considerable electrostatic contribution to the stability of the complex. Thus, with respect to the ESP surfaces at the active site, while annocatacin B retained the electrophilic pattern of the native active site of ND1, rotenone largely changed it to a nucleophilic one.An analysis of MM/PBSA showed that hydrophobic interactions were the main energetic component of the relative binding free energy (BFE), hence the important role of the aliphatic annocatacin B chains in their affinity for the ND1 subunit. Several favorable interactions were observed on these chains in the ND1—annocatacin B complex, including residues outside the active site (V113 and L266-L271), which allowed a 35% better energetic coupling than those observed in the ND1—rotenone complex. Despite the high structural fluctuations of the F223 residue in both complexes, a significant energy interaction was observed, favorable to the BFE (Our results suggest that the natural annocatacin B molecule could display better inhibitory capabilities than the rotenone molecule, an issue to be taken into account for future research.


PAP) plays a vital role in plant phosphate acquisition and utilization, as well as cell wall synthesis and redox reactions. In this study, comprehensive comparative analyses of PAP genes were carried out using the integration of phylogeny, chromosomal localization, intron/exon structural characteristics, and expression profiling. It was shown that the number of introns of the PAP genes, which were distributed unevenly on 12 chromosomes, ranged from 1 to 12. These findings pointed to the existence of complex structures. Phylogenetic analyses revealed that PAPs from tomato, rice, and Arabidopsis could be divided into three groups . It was assumed that the diversity of these PAP genes occurred before the monocot–dicot split. RNA-seq analysis revealed that most of the genes were expressed in all of the tissues analyzed, with the exception of SlPAP02, SlPAP11, and SlPAP14, which were not detected. It was also found that expression levels of most of the SlPAP gene family of members were changed under phosphorus stress conditions, suggesting potential functional diversification. The findings of this work will help us to achieve a better insight into the function of SlPAP genes in the future, as well as enhance our understanding of their evolutionary relationships in plants.Purple acid phosphatase ( Phosphorus (P), a key macronutrient, is required for plant growth and development. It not only plays an important role in energy transfer and metabolic regulation, but is also is a known important structural constituent of many biomolecules, including DNA, RNA, and protein . HoweverPAPs  [Purple acid phosphatases (PAPs) form part of the metallophosphoesterase family, which have been identified in some bacteria, plants, and animals . The resacteria) . PAP gene family have been identified in numerous plant species. For instance, in the model plant Arabidopsis, 29 different PAP genes were found to be annotated and the gene transcriptions of AtPAP11 and AtPAP12 were found to have been induced and increased during phosphate deprivation experiments [AtPAP10 is a Pi-starvation-induced phosphatase, is largely related with root surfaces, and plays a key role in plant resistance to phosphate limitations [AtPAP26 aid in scavenging P from organic P sources, it also plays a vital role in P remobilization [PvPAP3 was induced by Pi starvation in both the leaves and roots of common beans, and its expression levels were observed to be strictly dependent on two different conditions: phosphorus availability and the duration of the Pi-starvation conditions [PAPs also play important roles in both plant growth and development [At the present time, members of eriments , soybeanitations . Furtherlization . Previounditions . In addielopment ,21,22,23PAPs have been identified in tomato, two of which are secreted acid phosphatases (SAP1 and SAP2), and one of which is an intracellular acid phosphatase (IAP) [SAP1, SAP2, and IAP had different expression patterns. In a suspension cell system, the expressions of these two genes (SAP1 and SAP2) were specifically induced in response to Pi starvation. Meanwhile, the induction of IAP was observed on the sixth day of Pi starvation. It was observed that SAP1 was specifically expressed in the Pi-starved roots of 30-day-old seedlings, while IAP was expressed in the leaves, stems, and root tissue [SlPAP1, in tomato seedlings was cloned and characterized [PAP members in tomato, at present, the evolutionary relationships, expression patterns and potential functions of the members of PAP gene family in Pi acquisition and mobilization require further exploration. Previously, three Pi-starvation-responsive se (IAP) ,25. Howet tissue . Recentlcterized . AlthougPAP family genes in tomato were identified by integration of conserved motifs, gene structures, chromosome mapping, promoter cis-element and their phylogenetic relationships. These results will deepen our current understanding of the evolutionary relationships and functional divergence of the SlPAP genes in tomato, as well as provide some information for further comparative genomic studies throughout all of the plant family.At present, many gene families were identified widely in different plant whole genomes ,31,32,33PAP genes were identified and designated as SlPAP01 to SlPAP18, respectively, based on the gene chromosome locations. The information on all of the SlPAPs , isoelectric points (pI), numbers of exons/intros, and subcellular localization) are detailed in SlPAPs differed, ranging from 426 aa (SlPAP15) to 649 aa (SlPAP04), with an MW ranging from 47.9 kDa (SlPAP15) to 73.0 kDa (SlPAP04), and the pI varied from 5.80 (SlPAP15) to 8.22 (SlPAP21). The Euk-mPLoc 2.0 online tool was adopted to determine the subcellular localizations of the SlPAP proteins. All of the proteins were predicted to localize in the extracellular; four PAPs  were also localized in the cytomembrane and ER (SlPAP05 and SlPAP18) were observed to differ in those conserved domains of the PAPs. Furthermore, SlPAP05 lacked the first motif, and in SlPAP18, one amino acid residue in the second block was observed to have been altered. However, multiple amino acid sequence alignments showed the highest homology with the known plant PAPs, these two genes (SlPAP05 and SlPAP18) were still considered to be potential PAPs.In the present study, a total of 18 e and ER . Among tThe phylogenetic tree of the PAP proteins from tomato, Arabidopsis and rice is illustrated in This study also found seven pairs of orthologous genes between the various species: AtPAP1 and SlPAP06; OsPAP9b and SlPAP01; SlPAP05 and AtPAP12; OsPAP10a and AtPAP10; SlPAP16 and AtPAP15; AtPAP23 and OsPAP23; and SlPAP15 and AtPAP20, respectively. There were also determined to be nine pairs of paralogous genes among the species, four pairs from tomato, five pairs from rice, and nine pairs from Arabidopsis.SlPAP family genes were carried out by means of the online tool GSDS . The numbers of introns of the remaining SlPAP genes ranged from 1 to 12, as detailed in SlPAP01 and SlPAP06, which included 12 introns, and SlPAP04 contained lowest number. In addition, the SlPAP genes within the same subgroups were found to contain similar numbers of introns. Subgroup Ia had 7 to 9 introns; Subgroup Ib had 4 to 6 introns; Subgroup IIa had one intron; and Subgroup IIb contained 10 to 12 introns. In total, 10 distinct motifs were identified, and 139 motifs were identified in 18 SlPAP genes using MEME. Every gene contained 7 or 8 motifs, and all of the subgroups had motif 1, motif 2, motif 3 (with the exception of SlPAP01), motif 4, and motif 7  with the highest expression level had displayed tissue specificity in the roots, which was also observed in the S. pimpinellifolium L1589 variety  and reproductive organs (flower buds and flowers) between the two tomato genotypes were compared, differential expressions were observed between two genes (SlPAP12 and SlPAP18). In addition, there were differential expression profiles of four SlPAP genes in the vegetative and reproductive organs of tomato var. Heinz, while only one of those was expressed differentially in  variety A,B. The  variety A. It wasTo further deepen our current knowledge of the expression profiles of the SlPAP genes in different tissues, we analyzed the expression patterns of the SlPAP genes in the different tissues of the wild relative, S. pimpinellifolium C. Eight As shown in This study found that, by analyzing the expression profiles of the tandem duplications of SlPAP genes in the two genotypes and various types of tissue, that the two groups of SlPAP genes  had displayed a more similar expression pattern. Meanwhile, another group of SlPAP genes (SlPAP02/SlPAP03) was observed to display different expression patterns A,B. The In the present study, the respective expression levels of 18 SlPAP genes were performed under both low- and high-phosphorus conditions, so as to quantify their expression levels using qRT-PCR technology. The results showed that among the 18 SlPAP genes, 8 genes  were not detected in the control or treatments . The expArabidopsis had more interaction partners than others. All of the proteins involving molecular function  and cellular components  were detected by Gene Ontology. The metal ion binding (GO:0046872) and extracellular regions (GO:0005576) were detected in most of the proteins. To understand the interactions of the SlPAP proteins, the STRING website was used to construct an interaction network. The results showed that only 8 SlPAP proteins were selected because of their reliability . The SlPPAPs have diverse physiological functions, including phosphorus (P) acquisition [PAPs have mainly been studied for their potential involvement in Pi nutrition. These enzymes can catalyze the hydrolysis of various phosphate esters and anhydrides within the pH range from 4 to 7 [SlPAPs remain unclear at the genome level. Plant uisition ,34,35; cuisition ,37; the uisition ,39; respuisition ,41; invouisition ; and celuisition ,42,43. Im 4 to 7 . The majPAP genes in tomato using the tomato genome database  by bioinformatic method , when compared with Arabidopsis (genome size 125 Mb) and rice (480 Mb), the number of the PAP family (18) appears small. Among the 18 SlPAP genes, 1 predicted SlPAP gene was found to have incomplete conserved domains. However, the gene was still considered to be a potential PAP due to its overall amino acid sequences, which exhibited the greatest homology with the known plant PAPs during BLASTing in NCBI. The present study identified 18 c method . The PAPPAP (IAP) and secretory PAPs (SAP) according to the functions of the PAPs. IAP is mainly used for the reuse of intracellular phosphorus and the degradation of single nucleotides, phospholipids, and so on, thereby releasing inorganic phosphorus for the normal growth of plants [KbPAP is the first clear indication of the subcellular localization of plants due to its positioning in the cytoplasm [SlPAPs were only located in extracellular spaces , and the same subgroups exhibited similar intron/exon structures and motif patterns. For example, four members of the Ia-2 Subgroup had 7 introns; four members of the Ib-2 Subgroup had 4 introns; and motif 6 was characteristic of Subgroup IIb, which became an important basis for the subgroup classification. Previous related studies reported that AtPAP10, AtPAP12, AtPAP25 and AtPAP26 are very important in the phosphorus deficiency adaptation of Arabidopsis thaliana. In particular, it is believed that AtPAP25 may play a role in P-deficiency signaling as a phosphoprotein phosphatase [SlPAP02, SlPAP03, SlPAP05, SlPAP07, and SlPAP11 in the same subgroup may have similar functions. However, when combined with the quantitative expressions after P stress treatment, it was found that SlPAP05 and SlPAP11 were not expressed in either the control or the treatment samples. The expression levels of the other four genes decreased under low P stress conditions when compared with the control. Therefore, the function of tomato PAP genes could not be inferred only from the function of the Arabidopsis PAP genes and further research is required. This study’s results also revealed that the 18 sphatase ,14,47,48SlPAP genes were successfully predicted. The analyses results identified five classes of cis-elements, including light responses, as well as process-, environment-, plant-tissue-, and binding-site-related cis-elements. It was found that all of the SlPAP genes had more than one process-specific cis-element, including ABRE, CGTCA-motif, TGACG, GARE-motif, P-box and TATC-box, TCA-element, TGA-element and ERE. As previously stated, SA, JA, ET, and ABA play key roles in plant responses to both biotic and abiotic stress, as well as in the regulation of developmental processes [SlPAP genes with regard to biotic and abiotic stress responses.In the current investigation, based on PlantCARE online software, the cis-elements present in the promoter sequences of the rocesses . In addiSlPAP genes were observed in various tissue types and developmental stages. The results showed that the expressions of 15 SlPAP genes were tissue- and development-specific under normal growth conditions. It was considered that all of those tissue- and development-specific expressed genes likely play vital roles in the growth and development of tomato plants, and that their functions require further investigation. In addition, the expression levels of three genes  were high in all the investigated tissue types, which implied that they may be closely linked to the physiological and biochemical activities of tomato plants under normal growth conditions. Furthermore, two genes (SlPAP12 and SlPAP18) exhibited differential expression levels between the cultivated tomato and wild variety S. pimpinellifolium in the vegetative and reproductive organs. Those findings indicated that the interspecies divergence of gene expressions may have occurred, which could potentially lead to functional specialization. According to the RNA-Seq data, the expression patterns of the SlPAP genes, 18 SlPAP genes were evaluated under high- and low-phosphorus conditions by applying a real-time quantitative RT-PCR analysis method. In Arabidopsis, it was found that the transcript levels were reduced for AtPAP26 during phosphatase stress, whereas the AtPAP12 transcript levels correlated well with the relative levels of secreted AtPAP polypeptides [OsPAP10a were specifically induced by Pi-deficiency in the shoot and root tissues. However, OsPAP10c was induced in the roots, yet not in the shoots [AtPAP12, AtPAP26, OsPAP10a, OsPAP10c, and six SlPAP genes belong to the same group (Group Ia). In this study, SlPAP02, SlPAP03, SlPAP07, and SlPAP18 were also observed to be significantly down-regulated by the low phosphorus treatments when compared with the control. Therefore, the results suggested that those PAP genes may have similar functions to those in Arabidopsis and rice. In conclusion, a comprehensive analysis of SlPAP proteins in tomato was carried out though bioinformatic method. However, the function of tomato PAP genes under P stress condition will be explored in the future.In the present investigation, for the purpose of obtaining a greater insight into the expression patterns and putative functions of peptides ,50. A qRe shoots . AtPAP12http://solgenomics.net/, accessed on 4 January 2022). The local database of the tomato genome sequences was constructed by the Bioedit7.0 software. In order to retrieve all the members of the PAPs family in tomato, two methods were applied to identify the local database of the tomato genome sequences by means of the BLASTP algorithm. First, the local genome sequence database was searched according to all the members of the PAPs gene family in Arabidopsis thaliana, in order to obtain the homologs of PAP genes in tomato. Next, we used the Hidden Markov Model (HMM) profile of PAPs, which was from the Pfam database , as a query to retrieve this local tomato database. The putative genes that were obtained using the above technique were then further identified using the Pfam database. The conserved motif structures were then analyzed using a CDD method . Next, the molecular weight and isoelectric point of these SlPAP proteins were predicted using the ExPASy server . In the current survey, all of the tomato genome sequence data were downloaded from the following website . Then, conserved motif analyses of the SlPAP genes were performed using the following parameters: The MEME  was used to number the motifs. The structural feature of the SlPAPs were adopted by the online tool GSDS . In this study, the subcellular localization of the SlPAP proteins was predicted using the online Euk-mPLoc 2.0  and a neighbor-joining method featuring bootstrap replicates (1000) in MEGA 7.0 software. Additionally, a pairwise deletion method was applied to address any gaps or missing data in the sequences, then branch lengths were assigned via the pairwise calculations of genetic distances.Each amino acid sequence of the purple acid phosphatases in the tomato genome and SlPAP genes was determined from the SGN database. The respective positions of the SlPAP genes were draw on the chromosomes by implementing MapDraw V2.1 software. The duplication characteristics  were subsequently analyzed using the following web address: http://chibba.agtec.uga.edu/duplication/index/locus (accessed on 4 January 2022). The chromosomal location of the http://string-db.org, accessed on 4 January 2022) was used to analyze putative protein–protein interactions of PAP proteins. Eighteen SlPAP protein sequences were uploaded to the Multiple Proteins database, and “Organism” selected Arabidopsos thaliana to compare analysis. Then, using “search”, the highest-scoring proteins were used to generate the functional protein interaction network.The STRING v11.5 database  [The regions located 1000 bp upstream (promoter region) from each of the ry 2022) . http://ted.bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi, accessed on 4 January 2022) were used to analyze the expression profiles of the candidate SlPAP genes within different tissues of cultivated Heinz tomato plants (Solanum lycopersicum) and its wild relative, LA1589 (S.pimpinellifolium). The leaves were treated with various bacteria and PAMPs. Several tissues  were used in the cultivated tomato plants. The fruit samples included the 1 cm, 2 cm, 3 cm, mature green, breaker, and breaker + 10 fruit. For the tomato wild species (S. pimpinellifolium), 10 different tissues and organs were selected for analysis purposes, namely young/mature leaves, hypocotyl, roots, cotyledons, young flower buds, anthesis flowers, 10/20-day post anthesis (DPA) fruit, and 33 DPA fruit (ripening fruit). In addition, tissue-specific transcriptome profiling of LA1589 ovaries and fruit were selected, including Ovule 0 DPA, pericarp 0 DPA, pericarp 4 DPA fruit, placenta 0 DPA, placenta 4 DPA fruit, septum 0 DPA, septum 4 DPA fruit, embryo 4 DPA fruit, endosperm 4 DPA fruit, funiculus 4 DPA fruit, and seed coat 4 DPA fruit. The various tissues were treated with different bacteria. The PAMPs included mock bacteria 6h, DC3000 6h, Pseudomonas fluorescens 6h, Pseudomonas putida 6h, and Agrobacterium tumefaciens 6h. Then, expression analysis of the candidate SlPAP family genes was performed via MultiExperiment Viewer (MeV) software [In the present study, RNA-seq data which were obtained from the Tomato Functional Genomics Database  and 10 h dark period (18 to 20 °C) conditions. The irrigating solution reported previously was adopted. Three biological replicates for each treatment were included in the experiments. All of the samples were immediately frozen in liquid nitrogen, after which they were stored at −80 °C for RNA isolation. SlPAP genes were performed through qRT-PCR analysis. In addition, the encoding regions of the SlPAP genes were used to design primers based on using Primer 5.0 software. The transcription levels of all the SlPAP genes is listed in 2O, and 1 µL of the passive reference dye. The thermal conditions reported previously were adopted. Finally, the 2△△Ct method was used to calculate the relative gene expression values. The tomato housekeeping gene GAPDH was used as an endogenous control for the purpose of normalizing the samples [The total RNA isolation was performed from the treated leaves using Trizol  and was further treated with DNase I . Then, 1 µg of each of the treated RNA samples was reverse transcribed using PrimeScriptTM RT reagent Kit for qRCR . The primer information of the  samples . qRT-PCR


Limb-girdle muscle dystrophy (LGMD) is the fourth most common genetic cause of muscle weakness, with LGMD type 2A (LGMD2A) being one of the most common adult-onset muscular dystrophies presenting with limb-girdle weakness, while LGMD type 2B (LGMD2B) being the most common distal myopathy.This study includes two cases. The first case is a 13-year-old male, with no family history of similar symptoms, who presented with lower extremity weakness at the age of nine, starting with proximal weakness of the lower extremities, progressively involving the upper extremities. He had scapular winging and contracture of both Achilles tendons. The second case involves a 19-year-old male, with a distant family history of weakness, who presented with lower extremity weakness at the age of 10. He had distal myopathy, mainly as foot drop and atrophic gastrocnemii.In both cases, cardiac, intelligence, and bulbar function are spared. Electroneuromyography (ENMG) for both revealed myopathic process. Genetic testing results revealed calpain 3 (CAPN3) and dysferlin (DYSF) abnormality, confirming the diagnosis of LGMD2A and LGMD2B, respectively.This will be the first of its kind adequately documenting two of the most common LGMD subtype in our locale. Clinical phenomenology and preferential muscle involvement lead one to the gold standard genetic testing in heritable myopathies, which was well established in this report. Limb-girdle muscular dystrophy (LGMD), first described around the late 18th century, is now a spectrum of 30 subtypes that have diverse manifestations due to different genetic involvement and genotype-phenotype variation [LGMD is the fourth most common genetic cause of muscle weakness with an estimated minimum prevalence of one in 20,000 . LGMD tySimilar incidence was reported in China and Japan, with LGMD2A being the most common subtype, followed by LGMD2B ,8. LocalWe, therefore, present two genetically confirmed cases of the two most common LGMD subtypes, LGMD2A and LGMD2B, and furthermore discuss the clinical phenomenology for both cases, as this governs the diagnosis and subsequent management for such cases.Case 1A previously healthy Filipino male had his first symptom at 10 years old, which manifested as tiptoeing. He has no related comorbidities, and developmental milestones were at par with age. No other complaints were noted at this moment, and the patient can do activities of daily living. The weakness of lower extremities progressed, needing hand support for knee extension while going up the stairs. There was also difficulty standing from a seated position. Nevertheless, he can walk without assistance and continue his daily activities.At the age of 13, the patient had difficulty carrying heavy objects and was easily fatigued. There was already a decline in his ability to perform daily activities such as difficulty in grooming. These symptoms prompted consultation with a neurologist.Upon examination, he had atrophic proximal muscle groups, hypotonia, and hyporeflexia in the upper and lower extremities. Bilateral scapular winging was noted, and heel cords were tight. He exhibits generalized weakness; however, weakness was more prominent in the proximal muscle groups of the extremities. On standing from a seated position, Gower’s sign was demonstrated. There were long tract signs. The mental status and bulbar, sensory, autonomic, and other systemic functions were spared.The total creatine kinase (CK) of this patient was >3200 U/L. Other examination results were also normal, including thyroid function examination and 2D echocardiogram. While electroneuromyography (ENMG) showed myopathic process. Left biceps brachium needle muscle biopsy was performed; the findings, however, were nonspecific but consistent with dystrophic process Figure . GeneticCase 2A 19-year-old Filipino male presented with progressive weakness of bilateral lower extremities. Developmental milestones were at par with age, and he had no comorbidities. The symptoms started at the age of 11, with progressive weakness of both lower extremities characterized as difficulty in running and dragging of his feet. This was despite muscle manipulation, consultation with a physiatrist, and physical therapy.The patient was then referred to a pediatric neurologist at the age of 16 due to the persistence and progression of symptoms. CK and ENMG were requested. He was subsequently referred to a geneticist. Gene testing for dystrophin gene, however, was negative. At the age of 18, the patient noted a decrease of muscle mass in both gastrocnemius muscles, progressing proximally to both thighs.On examination, both lower extremities had decreased muscle tone, and there was hyporeflexia Figure . Marked There was a distant family history of language and motor delay and a similar presentation of weakness among distant relatives.Total CK was significantly elevated at >3200 U/L. Both ALT and AST were also elevated. Other examination results were also normal, including thyroid function examination and 2D echocardiogram, while ENMG showed myopathic process.While muscle biopsy was contemplated, the patient opted to forego the procedure to prioritize resources on genetic analysis, with the leading and pathognomonic clinical phenomenology in mind. Genetic analysis showed a homozygous pathogenic variant at dysferlin (DYSF) gene creating nonsense mutation at exon 42.LGMD2A, as seen in case 1, presents with proximal weakness and atrophy, with sparing of facial and neck muscles. The most common clinical presentation of this condition includes scapular winging and scoliosis. Achilles tendon contracture and other joint involvement are also common manifestations that can be prominently seen early at the start of the disease process. Rarely, patients may develop abdominal wall hernia as the consequence of the weakness of the external oblique muscles ,10. The Three phenotypes have been identified based on clinical manifestations, particularly the distribution of muscle weakness and the age of onset : 1) LeydThis patient may be classified under the Leyden-Mobius phenotype of LGMD2A, as it coincides with the onset in the first decade of life and initial and predominant lower extremity involvement.LGMD2A is secondary to mutations in the CAPN3 (15q15.1-q21.1) gene, located in the long arm of chromosome 15 at position 15.1-21.1. The c.550delA mutation was initially thought to be a mutational hot spot, as it is the most common allele in the Caucasian population and was also found to account for 75% of mutant alleles in European countries . ReportsThe genetic study of the patient in case 1 revealed two pathologies in both copies of strands of CAPN3. One pathology is a missense mutation of amino acids arginine to cysteine at position 49 (NM_000070.2(CAPN3):c.145C>T p.Arg49Cys) and has only three previous registrations of the same mutation, one of which is pathogenic with the diagnosis of LGMD2A, and two registries are likely pathogenic . The othLGMD2B, on the other hand, also known as dysferlinopathy, has two clinical phenotypes based on clinical manifestations, particularly the distribution of muscle weakness ,10: 1) LThis initially involves the gastrocnemius and is later manifested by the inability to do plantar flexion and the presence of calf wasting. Progressively, the hamstrings and hip flexors are involved, and it is described by difficulty in climbing the stairs or rising from the ground.In both phenotypes, the upper extremities will be involved at the latter stage. Muscles may show selective atrophy . Other rare manifestations include distal myopathy with anterior tibial onset (DMAT) and intense calf pains and tenderness, followed by muscle wasting, asymptomatic hypercreatine kinasemia, and camptocormia. Patients with this condition have symptom onset in the second decade of life .The patient in case 2 presented with calf pain, weakness, and atrophy of the lower extremities, with a predominance of atrophy of both gastrocnemii, with some level of selective wasting  gene, which is located at the short arm of chromosome 2 at position 13.2 .2 gene, ,13. MostBoth calpain and dysferlin are not expressed in the cardiac myocytes. Hence, cardiac involvement among patients with these mutated proteins is rare .Muscle histology, immunohistochemistry, and immunoblotting may be done. The pathologic features of LGMD diseases mostly are nonspecific and may vary. Most of them may include the presence of dystrophy, myopathy, neuropathy, inflammation, evidence of chronicity and disease progression, and signs of activity. Other findings include rimmed vacuoles and ragged, red, ring, and moth-eaten fibers .Currently, there is no treatment for LGMD diseases. Management is directed toward symptoms, surveillance of probable consequences, and most importantly improvement of quality of life .Both cases manifested their own specific clinical presentation and phenomenology, with some pathognomonic signs that may point to the diagnosis. Both cases were also subsequently confirmed by genetic testing. This highlights the importance of genetic testing as a confirmatory diagnosis for hereditary muscular diseases. With the preponderance of genetic development, the standard of diagnosis has shifted from biopsy and staining to genetic testing. Most importantly, routine staining of biopsied muscle yields nonspecific results. Other diagnostic modalities, such as immunostaining and immunoblotting, may be done. However, these serve as modalities that are second in line once genetic testing yields a negative result or if the latter is unavailable.In the Philippines, there is a big dilemma regarding the diagnosis, management, and exposure to such cases. Nevertheless, thorough history-taking, assessment of family history, and knowledge about the proper diagnostic tests to be done will help in properly localizing and eventually diagnosing LGMD. Precise diagnosis is necessary for prognostication and more precise management.


The characteristics of foulant in the cake layer and bulk suspended solids of a 10 L submerged anaerobic membrane bioreactor (AnMBR) used for treatment of palm oil mill effluent (POME) were investigated in this study. Three different organic loading rates (OLRs) were applied with prolonged sludge retention time throughout a long operation time (270 days). The organic foulant was characterized by biomass concentration and concentration of extracellular polymeric substances (EPS). The thicknesses of the cake layer and foulant were analyzed by confocal laser scanning microscopy and Fourier transform infrared spectroscopy. The membrane morphology and inorganic elements were analyzed by field emission scanning electron microscope coupled with energy dispersive X-ray spectrometer. Roughness of membrane was analyzed by atomic force microscopy. The results showed that the formation and accumulation of protein EPS in the cake layer was the key contributor to most of the fouling. The transmembrane pressure evolution showed that attachment, adsorption, and entrapment of protein EPS occurred in the membrane pores. In addition, the hydrophilic charge of proteins and polysaccharides influenced the adsorption mechanism. The composition of the feed (including hydroxyl group and fatty acid compounds) and microbial metabolic products (protein) significantly affected membrane fouling in the high-rate operation. To produce biogas from POME, anaerobic membrane bioreactors (AnMBRs) have been proposed for their high capacity and small footprint; however, membrane fouling, which causes permeate flux decline, is a substantial limitation of the technique [Alternative energy sources are widely promoted for sustainable development, including in wastewater treatment. The palm oil industry is one of the industries that can practice effective energy recovery from its waste and wastewater ,2. Palm echnique ,7,8. An echnique  and filtechnique ,11, amonechnique . Irreverechnique ,13. Irreechnique ,16,17,18echnique . High OLechnique . The effIn this study, a fabricated polysulfone (PSf) hollow fiber membrane was used for POME treatment by AnMBR. The cake layer and bulk suspension were characterized by FTIR, CLSM, roughness, and field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray spectrometer (EDS) to observe and evaluate the fouling under high-rate conditions to understand the fouling composition and mechanisms over a long operation period.POME and sludge samples were collected from a palm oil factory in Surat Thani, Thailand. The characteristics of POME, including pH, temperature, total COD (TCOD), soluble COD (SCOD), total solids (TS), volatile solids (VS), suspended solids (SS), and volatile suspended solids (VSS), were analyzed after acid fermentation to remove fat, oil, and grease (FOG) and large particles in wastewater . The POMThe inoculum sludge was analyzed following standard methods . The iniPolysulfone resin (19 wt%), polyvinylpyrrolidone K30 (2 wt%), and propylene glycol  were dissolved in N-Methyl-2-pyrrolidone (NMP) at approximately 70 °C for about 5 h to form a homogeneous dope solution. Then, the dope solution was transferred into a polymer dope tank and kept overnight at 40 °C to eliminate the air bubbles formed during stirring and pouring. The degassed dope solution was used to fabricate polysulfone hollow fiber membranes (PSf) through a dry–wet spinning process. Distilled water and tap water at room temperature were used as bore fluid and coagulant, respectively. The dope solution and bore fluid (water) were pressurized by nitrogen gas through a spinneret, with an outer tube diameter of 1.06 mm and inner tube diameter of 0.66 mm, to from a coagulation bath of water. After separation and solidification, the membranes were collected by a roller. The obtained membranes were immersed in water over 3 days to completely remove the NMP used in the membrane fabrication. Next, the membranes were immersed in 10% aqueous glycerine solution for 1 h to preserve the pore structure during drying. The fabricated membrane had a molecular weight cut-off (MWCO) of 67 kDa. The virgin membranes were characterized for morphology, chemical composition, and roughness by FESEM with EDS , FTIR , and atomic force microscope , respectively.2 in a 10 L reactor. The module was comprised of 65 membrane fibers, each 24 cm in length, and a fiber outside diameter of 0.1 mm. The fibers were potted in a PVC module with epoxy resin, and the module had a diameter of 0.6 cm. The PSf hollow fibers were fixed only at the bottom. The module was operated in an outside-in flow regime under a vacuum pressure in the range of 0.15–0.25 bar, which was supplied by a peristaltic pump . The TMP at the head of the module was measured by the vacuum pressure gauge, which had a similar set-up to a typical membrane bioreactor operation [3/d, respectively. At the end of each period, the membrane module was removed and chemically cleaned before the next period. After sludge removal, the fouled membrane from each period was collected for foulant characterization. The physical backwash was set up under 0.5 bar for 1 h followed by the chemical cleaning [A schematic diagram of the AnMBR setup is presented in peration ,20. The cleaning . The cheThe biomass sample from the AnMBR system was measured for its MLSS, MLVSS, and EPS concentrations. MLSS and MLVSS in the reactor were measured twice a week using standard methods . EPS solOrganic foulingThe membrane filtration was operated under subcritical flux in the TMP constant mode. The permeate flux and permeability rate were measured daily. To minimize physical fouling, gas sparging was added at 1.25 ± 0.25 L/hr through a gas recirculation from the AnMBR tank and internal liquid recirculation. At the end of each period, the foulants in the reactor and at the membrane surfaces were analyzed and characterized, as follows.Inorganic foulingThe hollow fiber membranes in the AnMBR were cut into small pieces of 1 cm length, and the biofilms (attached cells) were dyed with SYTO 9 for 30 min in the dark at room temperature, in order to analyze the distribution of the bacterial cells . Then, tThe membrane preparation was conducted following a procedure reported by Kaya et al. . The memThe average of MLSS concentrations during periods I, II, and III were 39.51 ± 2.49, 38.04 ± 1.12, and 36.71 ± 1.17 g/L, respectively. As the OLR in each subsequent period was increased by increasing the feed flow rate, higher concentrations of biomass in the reactor were achieved. In this experiment, a small fraction of biomass was lost as the membrane module was removed from the reactor for chemical cleaning between each period of the experiment. The average EPS concentration was around 166.02, 177.27, and 193.15 mg/L for periods I, II, and III, respectively. Protein made up a large fraction of EPS (77–79%), as shown in 2/h during periods I, II, and III, respectively  was nearly equal . Within The higher OLR induced an increase in F/M ratio b and a dCLSMAs seen in FTIR−1, 2964 cm−1,1584 cm−1, 1244 cm−1, 1115 cm−1, 832 cm−1, and 558 cm−1  and amide II functional group (peak 3) , which was related to the stretching vibration of C–O–C from glycosidic bonds, was found in fouled membranes and Zone C of the EPS.Summary of FTIR spectra peak assigned for the samples is showed in 558 cm−1 a. These (peak 3) a, which (peak 3) b. These proteins . The FTI870 cm−1 b. These surfaces . The preFESEMFESEM images of the top surface a and croRoughness of the MembraneThe surface probe micrograph (AFM) results indicated a typical morphology  for membranes . The top−1 and 1400 cm−1. The layer had a compact structure and was high in thickness of the protein portion. The increase of surface roughness reduced the membrane hydrophilicity (O-H stretching). The membrane hydrophobicity caused adhesion of protein molecules, adsorption at the membrane surface, and entrapment within membrane pores, respectively. The pore blocking mechanism occurred because of biofouling accumulation. The accumulation of microorganisms and cake layer increased but EPS products decreased due to the lower HRT. On the other hand, polysaccharides easily detached from the membrane as a result of their hydrophilic properties and the control of hydrodynamic conditions with internal recirculation. In addition, on the membrane surfaces, the cake layer was firmly attached, as was scaling by silica complexes combined with humic acid. The scaling on the fouled membrane was confirmed by EDX and FTIR results. These fouling mechanisms and the behavior of foulants can be considered as a progression ((1)2/h). After that, the cake layer materialized. The flux decreased and reached the local flux instead, causing the TMP to increase from 0.15 bar to 0.18 bar. The organic substances in the bulk feed were the major foulants on membrane surfaces. During this stage, the effect of foulant accumulation in the membrane pores (pore blocking) was minor.Stage I: initial fouling. The TMP slightly decreased over a short period during days 1 to 20, then a rapid increase of TMP occurred. During the initial decrease of TMP, the flux increased and approached the critical flux (2.8 L/m(2)Stage II: intermediate adsorption fouling. From day 20 to 70, the TMP remained constant at 0.20 bar. The cake layer was attached to the membrane surfaces, for which EPS, especially proteins, was adsorbed on the surfaces. A fraction of EPS was accumulated in the membrane pores and was entrapped there by the charge adsorption process.(3)Stage III: cake and pore blocking. Day 71 onwards, the TMP had risen to 0.25 bar. A dense cake layer accumulated at the surface, and pore blocking occurred simultaneously. In addition, adjacent to the cake layer, the bound EPS released and attached to the membrane.This study focused on evaluating fouling mechanisms and characterizing the foulants that occurred on a lab-scale AnMBR used to treat POME. We found that the predominant foulant was protein-EPS, which attached on the membrane surfaces through cake layer formation. The FTIR spectra of the cake layer on the fouled membranes showed peaks at 1638 cmgression . The corOverall, this study presented insight into the effect of operational parameters on fouling behavior in the lab-scale AnMBR during long-term operation. However, in order to commercialize AnMBR for POME treatment in a full-scale operation, the reported design and management results obtained need to be verified on larger-scale systems, and pre-treatment to remove suspended solid may be needed to mitigate membrane fouling.The growing cake layer, which resulted from high OLR and high MLSS, initiated biofilm formation on the membrane surfaces. The biofilm, in turn, bridged across the pores, resulting in increased TMP.EPS accumulated on the cake layer and, thus, plugged the membrane pores. The fouling from polysaccharide EPS can be mitigated by control of hydrodynamic conditions using internal recirculation. Due to the charge on the surfaces and the interaction between proteins and the membrane surface, the removal of protein EPS fouling was more difficult.The precipitation of inorganic compounds, silica, and phosphorus, also occurred in the AnMBR system. These compounds were then attached to the cake layer and caused membrane fouling.A high organic loading rate anaerobic membrane bioreactor was operated for 270 days (considered a long operation period). Fouling mechanisms were investigated, providing the following conclusion:Hence, in order for the lab-scale AnMBR to extend its operation time with high OLR, the two following conditions must occur: MLSS must be lower than 40 g/L and the internal recirculation must be higher than 1.25 L/d. High internal recirculation is needed to create violent hydrodynamic turbulence and minimize fouling. Furthermore, to improve the surface charge of the membrane , future research should focus on the modification of membrane surface properties to reduce the charge interaction between foulants and membrane surfaces.


Little is known about the differences in coronavirus disease (COVID-19) vaccine acceptance and hesitancy between the general population and healthcare workers in Japan. To compare these differences, a nationwide web-based cross-sectional survey was conducted on 19 January 2021, shortly before the initiation of COVID-19 vaccinations in Japan. A total of 6180 men and women aged 20–69 years and 1030 healthcare workers aged 20–69 years were enrolled. Data on COVID-19 vaccine acceptance, basic characteristics, including socioeconomic factors, and confidence in immunization in general were collected. COVID-19 vaccine acceptance was also evaluated under hypothetical vaccine effectiveness and adverse event frequencies. Factors associated with vaccine hesitancy were examined using multinomial logistic regression analysis. The COVID-19 vaccine acceptance rate was 48.6% among the general population and was lower among nurses (45.5%) and medical clerks (40.7%). Women and young adults had significantly higher COVID-19 vaccine hesitancy odds ratios, and current smokers had significantly lower odds ratios. The frequency of adverse events was a COVID-19 vaccine hesitancy factor. Even if these factors were adjusted, COVID-19 vaccine hesitancy among nurses was 1.4 times higher than that among the general population. Thus, interventions to improve health literacy and vaccine hesitancy among the general population and healthcare workers, especially nurses, are needed. Vaccine hesitancy is a challenge in promoting vaccination against coronavirus disease COVID-19). In 2015, the World Health Organization (WHO) Strategic Advisory Group of Experts on Immunization defined vaccine hesitancy as a delay in acceptance or refusal of vaccination despite the availability of vaccination services 9. In 201. A surveA pre-COVID-19 survey of more than 280,000 people in 149 countries and regions found Japan to have one of the lowest rates of vaccine confidence in the world . Thus, CThis study aimed to compare COVID-19 vaccine acceptance and hesitancy between the general population and healthcare workers in Japan.This was an Internet-based cross-sectional survey study conducted in Japan. The survey was conducted on 19 January 2021, at a time when the number of COVID-19 cases had significantly increased, and a state of emergency was declared for Tokyo, Chiba, Saitama, Kanagawa, Tochigi, Gifu, Aichi, Kyoto, Osaka, Hyogo, and Fukuoka.Study participants were registered on a panel of a web survey company . Panel membership was provided on a voluntary basis, and incentives were provided as points when the participants joined the survey. These points could be used to purchase products and services from partner companies. Approximately 1.2 million people were registered on this research panel.We calculated the sample size needed to investigate factors associated with vaccine hesitancy as follows: assuming α = 0.05/number of variables (50 items) = 0.001, β = 0.20, odds ratio = 1.5, vaccine hesitancy = 30–50%, and the proportion of associated factors was 10–20%, the required sample size was 2000–5000. We set 6000 men and women aged 20–79 years from the general population and 1000 healthcare workers aged 20–69 years as the target study population who were extracted from the membership panel. The survey was explained by e-mail, and study participants were enrolled using e-mails and apps until the target number was reached. The questionnaires were placed in a secure section of the website, and responders were required to answer each question so that there were no missing variables. Participants were considered to have consented by answering the survey questions. This study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Saga University, Saga, Japan (No: R2–24).COVID-19 vaccine acceptance and hesitancy were measured by how many respondents agreed with the following statement: “When a vaccine for COVID-19 becomes available, I will get vaccinated.” A five-point Likert-type rating scale was used with the following range of choices: “strongly disagree, disagree, neither agree nor disagree, agree, and strongly agree.” Those who chose “strongly disagree” and “disagree” were defined as hesitant. Those who chose “agree” and “strongly agree” were defined as accepting. Those who chose “neither agree nor disagree” were defined as “not sure.” The COVID-19 vaccine acceptance levels were also measured under the following hypothetical effectiveness and adverse event frequencies: effectiveness of protection against severe symptoms, 50%, 70%, and 90%; duration of vaccine effectiveness, 1 year, 3 years, and 5 years; serious adverse events, such as death or hospitalization, occurrence in one in 10,000 doses, one in 100,000 doses, and one in 1,000,000 doses; and mild adverse events, such as flu-like symptoms or high fever, occurrence in one in two doses, one in five doses, and one in ten doses.Vaccine confidence is defined as trust in the effectiveness and safety of vaccines and the healthcare system that delivers them . In thisParticipants chose all of the factors that applied to them as important factors in deciding whether or not to vaccinate in listed factors in follows: age, comorbidity, occupation vaccination fee, doctors’ recommendation, vaccine effectiveness, duration of effectiveness, frequency of adverse events, family recommendation, and the COVID-19 epidemic situation.Data on the following sociodemographic factors were collected: sex, age group , occupation, state of emergency in their residential area, marital status (married or not), have children or not, household annual income category , highest level of education, current smoking status, height, weight, comorbidities , and influenza vaccination status in the previous (2019/20 season) and present season (2020/21 season).2 test was performed to test the differences between the general population and healthcare workers. A multinomial logistic regression analysis was performed to test the factors associated with COVID-19 vaccine hesitancy, with reference to the acceptance group. Among the significant factors identified by univariate analysis, those factors that were strongly correlated with one another, and were more compatible with the model, were used as explanatory variables in the multivariate analysis. Age- and sex-adjusted scores for vaccine confidence and literacy were analyzed using multiple regression analysis. Two-tailed tests were performed at a significance level of <5%. SAS version 9.4  was used for statistical analysis.A χA total of 6180 men and women aged between 20 and 69 years (515 men and women per 10-year age group) and 1030 healthcare professionals participated in this study. A comparison of the characteristics of the general population and healthcare workers is presented in Regarding COVID-19 vaccine acceptance and hesitancy, the distribution of responses  to the following two questions was similar between the groups: “When a vaccine for COVID-19 becomes available, I will get vaccinated” and “When everyone gets a vaccine for COVID-19, I will get vaccinated” . The COVFactors associated with “hesitant” and “not sure” responses regarding COVID-19 vaccination are shown in COVID-19 vaccine acceptance was also compared between the general population and the healthcare workers under hypothetical vaccine effectiveness, duration of effectiveness, and adverse event frequencies . COVID-1As shown in This was the first study to compare COVID-19 vaccine acceptance and hesitancy between the general population and healthcare workers in Japan. We found that nurses are 1.4 times more likely to be hesitant to the COVID-19 vaccine as compared to the general population.Previous studies have reported COVID-19 vaccine acceptance rates of approximately 70% in the United States , 80% in Several web-based cross-sectional surveys on COVID-19 vaccine acceptance and hesitancy have been reported in Japan ,13,14,23Female sex, younger age, being a nurse, influenza vaccine hesitancy, and concern about the frequency of adverse events were positively associated with vaccine hesitancy in our study. These factors have often been reported in previous studies ,18. SmokThe vaccine acceptance under hypothetical vaccine effectiveness and adverse events was previously examined in the U.S.  and JapaThe strength of the present study is that we compared vaccine acceptance and hesitancy between the general population and healthcare workers who are influential regarding vaccine acceptance in the general population. We must acknowledge that this study had several limitations. First, web-based surveys tend to have selection and sampling biases. The survey participants may comprise regular Internet users; thus, they might have a higher socioeconomic status, which influences vaccine perception. However, most surveys on COVID-19 vaccine perception have used the same method; thus, our results are comparable to those of other studies. In addition, the degree of interest in the COVID-19 vaccine might not have influenced participation because participants received points, which could be used to purchase products and services from partner companies after completing the survey. Second, this was a cross-sectional study; therefore, causal relationships could not be established. However, our purpose was not to establish causality but to assess the factors associated with vaccine acceptance and hesitancy. Lastly, this study was conducted before the initiation of COVID-19 vaccinations in the population, and vaccine perception may change over time. In addition, the survey was conducted prior to the Delta variant outbreak in Japan, which may propel people to become vaccinated because it is highly contagious; therefore, the time at which the survey was conducted is an important consideration. Despite these limitations, to the best of our knowledge, this is the first study to compare COVID-19 vaccine acceptance and hesitancy and their related factors between the general population and healthcare workers in Japan.The COVID-19 vaccine acceptance rate was 48.6%, and the hesitancy rate was 17.5% among the Japanese general population in January 2021. Nurses showed lower COVID-19 vaccine acceptance and higher hesitancy. Similar to previous studies, a higher proportion of women and young people, those who did not receive the influenza vaccine, and those who cited the frequency of adverse reactions as a factor influencing their decision on vaccination showed vaccination hesitancy. Even after adjusting for the influence of these factors, nurses are 1.4 times more likely to be hesitant as compared to the general population. Confidence in immunization in general was higher among healthcare workers than among the general population. However, safety concerns were greater among healthcare workers. Interventions to improve immunization literacy are needed among both the general population and healthcare workers.