Most of the high-grade serous ovarian cancers (HGSOC) are accompanied by P53 mutations, which are related to the nucleotide excision repair (NER) pathway. This study aims to construct a risk signature based on NER-related genes that could effectively predict the prognosis for advanced patients with HGSOC. In our study, we found that two clusters of HGSOC with significantly different overall survival (OS) were identified by consensus clustering and principal component analysis (PCA). Then, a 7-gene risk signature  for OS prediction was developed subsequently based on TCGA cohort, and the risk score-based signature was identified as an independent prognostic indicator for HGSOC. According to the risk score, HGSOC patients were divided into high-risk group and low-risk group, in which the distinct OS and the predictive power were also successfully verified in the GEO validation sets. Then we constructed a nomogram, including the risk signature and clinical-related risk factors (age and treatment response) that predicted an individual’s risk of OS, which can be validated by assessing calibration curves. Furthermore, GSEA showed that the genes in the high-risk group were significantly enriched in cancer-related pathways, such as “MAPK signaling pathway”, “mTOR signaling pathway”, “VEGF signaling pathway” and so on. In conclusion, our study has developed a robust NER-related genes-based molecular signature for prognosis prediction, and the nomogram could be used as a convenient tool for OS evaluation and guidance of therapeutic strategies in advanced patients with HGSOC. High-grade serous ovarian cancer (HGSOC) is of great concern to the researchers among all ovarian cancers, as it accounts for 70%–80% of deaths from ovarian cancer. The modes of carcinogenesis, molecular-genetic characteristics, and the origin are distinctive from low-grade serous ovarian cancer . Due to In recent years, genome-wide expression profiling detection can effectively provide detailed information for the prognosis assessment of cancer patients . In breaDNA damage response and repair pathways play an essential regulatory role in the occurrence and development of ovarian cancer . DNA repIn the present study, we comprehensively explored the roles of 31 NER-related genes in HGSOC based on multiple transcriptome datasets, such as The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) Project, and the Gene Expression Omnibus (GEO) database. We evaluated the interaction and correlation among the 31 NER-related genes and employed the consensus cluster analysis to identify two HGSOC clusters with different clinical outcomes based on their expression patterns of these genes. Then we conducted the least absolute shrinkage and selection operator (LASSO) Cox regression to obtain a 7-gene signature on TCGA HGSOC cohort. This robust risk signature was successfully confirmed in two GEO validation sets and showed an excellent predictive effect on prognosis. Moreover, the risk signature and clinical characteristics were used to construct a nomogram to predict the prognosis of advanced patients with HGSOC. Finally, we also used the gene set enrichment analysis (GSEA) to explore the differences in the signaling pathways between subgroups classified by risk signature. Our results indicated that the risk signature derived from seven NER-related genes could serve as novel prognostic biomarkers for advanced patients with HGSOC.https://xenabrowser.net/datapages) (http://www.genenames.org)  (The study design flowchart is presented in tapages) . Accordimes.org) , and theect.org) .https://www.ncbi.nlm.nih.gov/geo). Gene expression in GSE13876 was performed using Operon human v3 ∼35 K 70-mer two-color oligonucleotide microarrays ; Gene expression in GSE49997 was performed using ABI Human Genome Survey Microarray Version 2 ; Gene expression in GSE17260 was performed using Agilent-014850 Whole Human Genome Microarray 4 × 44K G4112F (Probe Name version) ; Gene expression in GSE63885 was performed using Affymetrix Human Genome U133 Plus 2.0 Array . Then, we integrated 346 standards-compliant samples from three datasets  as a combined validation set based on the exclusive criteria to improve the sample size, and we executed batch normalization between these three platforms using the “sav” and “limma” packages in R to avoid generating unreliable results  were downloaded from Gene Expression Omnibus (GEO) database  for gene screening. A total of 8,466 common genes were selected. According to previous studies , 31 sharp < 0.05. The mRNA expression profiles of 31 NER-related genes were obtained. The heatmap and violin plots were presented by “pheatmap” and “ggplot2” packages in R.We first analyzed the mRNA expression levels between HGSOC tissues and normal tissues by using the “limma” package with cut-off criteria of via the Search Tool for the Retrieval of Interacting Genes (STRING) (https://string-db.org/) (All 31 NER-related genes were used for the protein-protein interaction (PPI) analysis with a combined confidence score  ≥ 0.9 db.org/) . PearsonConsensusClusterPlus” package in R  was applied to explore the clinical implications of the 31 NER-related genes in the TCGA HGSOC cohort. The number of clusters and their stability were determined by the consensus clustering algorithm  were determined by the univariate Cox regression analysis of 31 NER-related genes; the hazard ratios (HRs) of genes <1 or >1 were regarded as protective or risk genes, respectively. To prevent over-fitting in our analysis, the least absolute shrinkage and selection operator (LASSO) regression analysis was used to identify the optimal prognostic model out of the selected seven candidate genes  database.Similarly, the risk score of each advanced patient with HGSOC from the GSE13876 and the merged GEO datasets was calculated based on the formula above. Taking the median risk score in the TCGA cohort as the cut-off value, HGSOC patients in both validation sets were divided into high- or low- groups. The K-M method and log-rank test were employed to calculate OS with an overall significance level of p-value < 0.05, and false discovery rate (FDR) q-value < 0.25 were significantly enriched.Gene set enrichment analysis (GSEA) was used in the TCGA cohort to investigate the potential biological pathways underlying the different risk groups defined by the 7-gene expression signature. Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets (v7.1) and phenotype label (high risk vs. low risk) files were generated and loaded into the GSEA software . The permutation test run 1,000 times. The pathways with normalized enrichment score (NSE) absolute value >1, normalized limma”, “sva”, “ConsensusClusterPlus”, “pheatmap”, “ggplot2”, “corrplot”, “pROC”, “rms”, “survival” packages were used for analysis or visualization in R. All statistical tests were two-sided and p < 0.05 was considered statistically significant.Unless otherwise specified, all statistics analyses were performed with R software. The differences between groups for the continuous and categorical variables were respectively assessed by studentʼs t-test or one-way ANOVA and the Chi-square test. The “To better understand the importance of the NER-related genes in tumor initiation and progression, we firstly investigated expression levels of NER-related genes in different tissue samples of the merged TCGA-GTEx dataset. The TCGA-GTEx cohort comprised 326 advanced patients with HGSOC and 88 ovaries of healthy donors. The clinicopathological characteristics of the TCGA HGSOC cohort were listed in The interaction relationships among the 31 NER-related genes were shown by the PPI network, and the number of interactions for each gene was counted in p = 0.021), which suggested that the 31 NER-related genes could classify the advanced patients with HGSOC at the prognostic level (p > 0.05).Next, TCGA HGSOC samples were selected for the subsequent consensus clustering analysis. According to the expression similarity of the 31 NER-related genes, k = 2 could be the optimal choice when clustering stability datasets increased from k = 2–9 . We notiic level . We thenp < 0.1). Among these seven genes, only DDB2 and POLR2D were considered as protective genes with HR <1, while RPA2, CCNH, XPC, ERCC2, and ERCC4 were considered as risky genes with HR >1  (p = 0.049), there were no significant differences between the high- and low-risk groups in the age, grade, stage, and residual tumor .According to our previous results, the collinearity between these genes may affect the accuracy of traditional Cox regression analysis . Therefo707e-05) . The 5-y707e-05) . Next, t707e-05) . The resMoreover, the Human Protein Atlas (HPA) database was used to validate the cellular sub-localization and expression patterns of the seven selected genes in serous ovarian cancer tissues and normal ovarian tissues at the protein levels . HPA anan = 415) and the merged GEO dataset (n = 346), respectively. According to the median risk score as the cut-off value in the TCGA HGSOC cohort, 192 (46.3%) patients were classified as low-risk, and 223 (53.7%) as high-risk in GSE13876; 255 (73.7%) patients were classified as low-risk, and 91 (26.3%) as high-risk in the merged GEO datasets. The corresponding 5-year OS was 24.1% for the high-risk group and 35.0% for the low-risk group in GSE13876 , or patients with CR/PR (p = 1.214e-03), or patients with optimal cytoreductive surgery (p = 2.263e-03), or those with non-optimal cytoreductive surgery (p = 0.025). However, no significant difference was observed for OS between high- and low-risk groups for patients with FIGO stage IV (p = 0.116), or patients with SD/PD (p = 0.245) due to sample size limitation.To confirm that the NER-related genes based on classifier had similar prognostic value in different cohorts, we assessed the samples in GSE13876 (p = 0.077), a nomogram that combined the age, treatment response, and risk signature was developed to predict 3- or 5-year survival of advanced patients with HGSOC , inositol phosphate metabolism , phosphatidylinositol signaling system , MAPK signaling pathway , and VEGF signaling pathway . In contrast, two downregulated biological processes/signaling pathways were observed in the high-risk group: DNA replication  and spliceosome   . These rSerous ovarian cancer was divided into low-grade serous ovarian cancer and HGSOC, and they had significantly different features in genomics, clinical manifestations, origin, and prognosis . The preNER is a complex biochemical process that requires multiple proteins assemble in an ordered at base damaged sites and then function as a multi-protein complex . In our We also identified a risk signature of seven genes consisting of ERCC2, ERCC4, POLR2D, DDB2, XPC, CCNH, and RPA2 that predicts OS in the TCGA and GEO datasets. ERCC2 single nucleotide polymorphisms (SNPs) were found to be associated with an increased risk of ovarian cancer . The samThe mechanisms were further investigated to reveal the causes of the different prognosis of the two HGSOC risk subgroups stratified by the 7-gene signature. GSEA demonstrated that some pivotal signaling pathways and biological processes were significantly enriched in the high-risk group with poor survival, including inositol phosphate metabolism, MAPK signaling pathway, mTOR signaling pathway, phosphatidylinositol signaling system, and VEGF signaling pathway. It was reported that inositol phosphate recycling regulated glycolytic and lipid metabolism that drove cancer aggressiveness . It was Despite encouraging findings in the present study, several limitations still exist. First of all, our results are mainly based on bioinformatics analysis. Although there are multiple datasets for mutual verification, experimental and clinical data will be needed to verify our results in the future. Secondly, although the risk signature and nomogram showed good prediction accuracy in the internal verification, their performance is still warranted validation in different HGSOC populations. Finally, our study did not contain clinicopathological information such as the scope of surgical resection and specific chemotherapy drugs, since TCGA did not cover such information, and the treatment standard for advanced patients with HGSOC has been controversial.Taken together, we profiled the sharply altered NER-related genes between HGSOC and normal samples, which may play a vital role in the progression of HGSOC. More importantly, a robust risk signature that was significantly associated with the clinical outcome of HGSOC was constructed and validated in two different GEO validation sets. In addition, we also developed a 7-gene nomogram containing the risk signature and clinical-related risk factors, which may aid the individualized prediction of the prognosis of advanced patients with HGSOC. Finally, further research on these genes may provide new insights into the potential relationship between the NER repair pathway and HGSOC prognosis.


TM cell analyzer revealed cell cycle arrest at G2/M phase and apoptosis induction. Inhibition of Bcl-2 and activation of Bax, leading to induction of the downstream caspase-dependent death pathway were confirmed by Western blot analysis. Co-culture with activated macrophages, kaffir lime extract and its constituents enhanced the development of pro-inflammatory (M1) macrophages and boosted TNF-α production, resulting in SCC15 apoptosis. Findings revealed novel potential activities of kaffir lime leaf extracts and their constituents in inducing M1 polarization against SCC15, as well as direct anti-proliferative activity.Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. Late-stage patients have a significant chance of local recurrence and distant metastasis, as well as poor prognosis. Therapeutic goals for patients must be improved and personalized to reduce adverse effects. This study explored the anti-proliferative activity and immunomodulation potential of the constituents of crude kaffir lime leaf extract  under co-culture. Results showed high cytotoxicity to human SCC15 cell line but not to human monocyte-derived macrophages. Treatment with crude extract and the contained compounds also suppressed cell migration and colony formation of SCC15 compared to the untreated control group, while high levels of intracellular ROS production were detected in the treatment group of SCC15. The Muse Head and neck squamous cell carcinoma (HNSCC) was the seventh most common cancer worldwide in 2018 with 890,000 new cases and 450,000 deaths .Cancer is a complicated disease involving tumor microenvironment (TME) that defines the nature of cancer, not by the genetics of the tumor cells alone but by the surrounding environment that the tumor cells need for survival, growth, proliferation, invasion, and metastasis. The production of numerous intercellular mediators such as cytokines, chemokines and vesicles attract non-transformed cells in the surrounding area. The consequences are TME formation and close interaction with cancer cells to support the development of cancer hallmarks –6. The TMacrophages play an important role in the innate immune system by maintaining immune homeostasis. They are involved in several processes of initiating and regulating the immune responses to foreign antigens . Two majPouteria campechiana leaves stimulated the proliferation of murine macrophages, phagocytic activity, nitric oxide (NO), hydrogen peroxide (H2O2) and cytokines (interleukin (IL)-6 and tumor necrosis factor (TNF)-α) production [Curcuma xanthorrhiza Roxb. significantly increased phagocytosis and release of NO, H2O2, TNF-α and PGE2 in a dose-dependent manner in macrophages by specific activation of nuclear factor-κB (Nf-κB) [et al. (2002) revealed that polysaccharides from safflower petals (Carthamus tinctorius L.) activated macrophages by recognizing and binding to the specific Toll-like receptor 4 on their surfaces [et al. investigated the immunomodulatory effects of Clinacanthus nutans extracts on THP-1 macrophages in co-culture with triple-negative breast cancer cells (MDA-MB-231). They concluded that this plant limited the pro-inflammatory condition in TME and showed potential anti-cancer properties on highly metastatic breast cancer condition [Macrophages are involved in tumor development and suppression. Immunostimulation of macrophages contributes to tumor regulation as a promising therapeutic target. Numerous studies have revealed that many plants or natural compounds have an immunomodulatory effect on macrophages. Methanolic extract from oduction . Crude p (Nf-κB) . In Japasurfaces . Moreoveondition .Citrus hystrix DC., is commonly called makrut lime or kaffir lime. This plant belongs to the subfamily Aurantioideae, order Sapindales, family Rutaceae [Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae [Rutaceae . Kaffir Rutaceae . KL contRutaceae . VariousRutaceae . The leaRutaceae . KL exhifluenzae ,24. In afluenzae  and showfluenzae ,27. Our fluenzae .However, the anti-proliferative potential on head and neck cancer and the immunomodulatory effect of KL on macrophages remain unknown. Here, the anti-proliferative properties of KL and its active compounds on squamous cell carcinoma 15 cell line (SCC15) and the immunomodulatory effect on THP-1 derived macrophages against SCC15 were investigated.Powdered kaffir lime leaves Lot. No. 250818 were obtained from Khaolaor Laboratories Co., Ltd., Samut Prakan, Thailand. The method for KL extraction was described in our previous study . The powTM . Cells were incubated at 37°C with 5% CO2 for 7 days until fully differentiate to macrophages, with media replacement every 3 days.Human monocyte-derived macrophages (MDM) were used as the primary normal cell control. Buffy coat was obtained from the Blood Bank, Naresuan University Hospital, Phitsanulok, Thailand. Ethics approval was obtained from the Human Ethics Committee of Naresuan University (IRB no. P1-0127/2565). Buffy coat was diluted with Hank’s balanced salt solution (HBSS) and then overlaid on 5 ml Lymphoprep  and centrifuged at 2000 rpm for 30 minutes. The mononuclear cell layer was collected and washed twice with HBSS buffer and the peripheral blood mononuclear cells (PBMC) were suspended in 5 ml of RPMI medium. Then, monocytes were separated by size sedimentation centrifugation using Percoll  and the PBMC suspension was carefully overlaid on 10 ml of Percoll solution and centrifuged for 15 minutes at 2100 rpm. Monocytes between Percoll were collected and washed with HBSS, followed by centrifugation for 10 minutes at 1300 rpm. Isolated monocytes were cultured in RPMI 1640 and supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic purchased from Gibco2, with medium renewal every 2–3 days. The control conditions used untreated cells and Cisplatin  as an anti-cancer positive condition.Squamous cell carcinoma (SCC) 15 (ATCC® CRL-1623™) was purchased from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium and Ham’s F12 medium  containing 10% fetal bovine serum. Cells were incubated at 37°C with 5% CO2 for a further 2 days. THP-1-derived macrophages were cultivated with 20 μg/ml of lupeol, citronellal and citronellol and 100 μg/ml of crude hexane, and crude ethanol before being incubated for 24 hours. The phenotype of THP-1- derived macrophages was investigated by flow cytometry.THP-1 is a monocytic human leukemia cell line from the ATCC that can be differentiated into macrophages. This makes it possible to investigate, in co-cultures without direct cell contact interactions, how KL extracts and their constituents affect macrophage defense against cancer cells. THP-1 cells were maintained in RPMI 1640 Complete Medium supplemented with 10% FBS and 1% Antibiotic-Antimycotic . Differentiation of the THP-1 cell line was induced by stimulation with 10 ng/ml of phorbol-12-myristate-13-acetate (PMA)  containing RPMI for 3 days, and the cells were incubated in fresh complete RPMI 1640 at 37°C with 5% CO4 cells/50 μl/well. Cells were treated with various concentrations of compounds, lupeol, citronellol and citronellal . Moreover, cells were treated with serial dilutions of two crude extracts, KL hexane and KL ET at 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, and 10,000 μg/ml. Cells were then incubated at 37°C for 24 hours. Fifty microliters of MTT (0.5 mg/ml)  in medium free serum were added and cells were incubated at 37°C for 3 hours. MTT reagent was removed, and formazan crystals were dissolved in 100 μl of DMSO. Absorbance of the formazan solution was measured at 590 nm by a microplate reader . This method followed the previous protocol [MTT assay was used to determine the growth inhibitory role of crude extract and active compounds. MDM, THP-1-derived macrophage, and SCC15 were seeded in 96-well plates at a density of 1x10protocol . The hal4 cells/well and incubated for 24 hours. The cells were treated with extracts and compounds for 24 hours. The SCC15 cell line from each experimental condition was harvested using trypsin/EDTA solution  and incubated at 37°C for 5 minutes. Then 200 μl of completed DMEM HamF12 was added to stop the reaction of trypsin. Cells were aspirated and centrifuged at 1500 rpm for 5 minutes and then fixed with 70% ethanol and incubated for at least 3 hours at -20°C. Cells were then washed twice with cold PBS and resuspended in 200 μl of Muse™ Cell cycle reagent before mixing gently and incubating for 30 minutes at room temperature in the dark. Cell cycle stage was then analyzed by a Muse™ Cell Analyzer.To confirm the growth inhibitory role of KL H, KL ET, lupeol, citronellol and citronellal, the cell cycle was analyzed using Muse™ Cell Cycle Kit  following the manufacturer’s protocol. SCC15 cells were seeded in 24-well plates at a density of 1x10Muse™ Annexin V & Dead Cell Kit  was used for apoptosis study. SCC15 cells from each experimental condition were harvested by trypsin/EDTA solution as described in cell cycle assay. Cells were washed in PBS and resuspended in the medium with 1% FBS. Then 100 μl of Muse™ Annexin V & Dead Cell Reagent was added, mixed gently and incubated for 20 minutes at room temperature in the dark. Cell apoptosis was measured using a Muse™ Cell Analyzer following the manufacturer’s protocol.Colony formation assay was used to study the potentiality of single cells to form colonies. This assay followed the previous description . The SCC6 cells/ml and incubated at 37°C until reaching 80% confluence as a monolayer. The cell monolayer was scraped in a straight line with a SPL scar scratcher . Detached cells were removed and washed twice with 1 ml of medium. Cells were treated as described in the colony formation assay and incubated for 36 hours. A snapshot of cells from each condition was taken at time points of 6, 12, 24 and 36 hours using an inverted microscope . Distance of the wound area was analyzed by the ImageJ system.Cell migration of the SCC15 cell line was evaluated by wound closure assay modified from a previous method . The SCC2DCFDA  was used for detection of reactive oxygen species (ROS) production. Cells were seeded into dark clear bottom 96-well plates at 2.5 x 103 cells/well and incubated for 24 hours in standard culture conditions at 37°C. The medium was removed, and the cells were incubated with diluted H2DCFDA solution (20 μM) for 45 minutes at 37°C in the dark. Next, cells were washed with PBS and treated with cisplatin, KL H, KL ET, lupeol, citronellol and citronellal for 3 hours. The plate was measured immediately on a fluorescence plate reader at Ex/Em = 485/535 nm in end point mode.HSCC15 were cultured with 12.5 μg/ml of cisplatin as a positive control. Cells were treated with each compound , crude hexane and crude ethanol at 100 μg/ml. Total proteins of the SCC15 cell line from each condition were extracted by ice-cold RIPA lysis buffer  in the presence of Halt Protease/Phosphatase Inhibitor Cocktails , and centrifuged at 12,000 rpm for 15 minutes at 4°C. Quantification of total protein concentration was performed by the Bradford Coomassie-binding colorimetric method. The protein extract was mixed with an equal volume of 4X Leammli loading buffer and heated to denature at 95°C for 5 minutes. Samples were loaded into wells of 12% SDS-polyacrylamide gel electrophoresis (PAGE), and proteins were separated according to molecular weight and transferred to a polyvinylidene fluoride membrane . For Western blot analysis, the membrane was blocked overnight at 4°C with blocking buffer containing 5% bovine serum albumin  in Tris-buffered saline with Tween 20 (TBST) buffer. The membrane was blotted using primary antibodies specific to cleaved-caspase 3  , β-actin, pro-caspase 3, Bax and Bcl-2  for one hour at room temperature on a rotator. The membrane was then washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) secondary antibody  for one hour at room temperature. The membrane was observed by soaking in chemiluminescence substrate for 5 minutes and placed in a ChemiDoc XRS+ Imaging System . The chemiluminescence signal of the blotted membrane was detected by Image Studio Lite software .Cell surface markers were analyzed by immunostaining with PE-conjugated anti-human CD80 and FITC-conjugated anti-human CD163 . Cells were suspended with FACS buffer solution and directly stained with fluorescence-conjugated antibody. The reaction was incubated at 4°C for 1 hour in the dark and the cells were then washed twice with PBS. The cell pellet was resuspended with staining buffer and analyzed by an FC 500 Flow Cytometer . FITC-conjugated mouse IgG1,κ isotype control and PE-conjugated mouse IgG1,κ isotype control  were used as the negative control.4 cells/well) were seeded into the upper chamber of the transwell apparatus. THP-1 monocytes were differentiated into macrophages by 3 days of incubation with 10 ng/ml of phorbol 12-myristate 13-acetate . Cells were then washed, and the fresh complete RPMI was replaced for a further 2 days. THP-1-derived macrophages were cultivated with 20 μg/ml of lupeol, citronellal and citronellol and 100 μg/ml of crude hexane, and crude ethanol. SCC15 were seeded in the lower well at 5 × 104 cells/well for 24 hours to allow their adherence to the walls. The chambers with the THP-1-derived macrophages were washed and then placed directly on top of the well plates containing the SCC15 cells, and the resulting co-culture systems were incubated for 24 hours. SCC15 was collected for cell apoptosis analysis using Muse™ Cell Analyzer.In the co-culture experiments, THP-1 monocytes were differentiated in 24-transwell inserts . The THP-1 monocytes . A 96-well plate was coated with capture antibody specific to TNF-α at 4°C for one night. The plate was washed four times with a wash buffer between each step. The plate was then blocked with blocking solution for 1 hour with shaking. Supernatant from the cell culture of each condition and the standard were placed into the reaction and shaken for 2 hours at room temperature. The plate was washed, followed by binding of 100 μL of detection antibody, and incubated for 1 hour before washing. Avidin horseradish peroxidase-conjugated secondary antibody was then added, and the plate was shaken for 30 minutes. The reaction was completed by adding 100 μL of freshly mixed solution of 3,3’,5,5’-tetramethylbenzidine (TMB). After incubating for 15 minutes in the dark, the stop solution was added. The absorbance of the reaction was measured at 450 nm using an EnSpire® Multimode microplate reader . The production of cytokines in each condition was calculated from standard curves using known concentrations of recombinant cytokines.p = 0.05) was used in all statistical analyses.All experimental conditions were triplicated to provide accurate results. One-way ANOVA and the Bonferroni multiple comparisons test were used for data analysis by GraphPad Prism software. A confidence interval of 95%  and active compounds . Human monocyte-derived macrophages (MDM), SCC15 and THP-1 were treated with different concentrations of extracts and compounds for 24 hours. IC5 values of lupeol, citronellal and citronellol on human MDM were 112 μg/ml, 171.5 μg/ml and 98.5 μg/ml, respectively  and highCell cycle assay was assessed by a Muse™ Cell Cycle Kit and nuclear DNA of the cell line was intercalated with propidium iodide (PI). Cells were discriminated at different phases of the cell cycle based on differential DNA content including G0/G1, S and G2/M phase. To investigate the effect of the extracts, lupeol, citronellal and citronellol on cell cycle progression, untreated and treated SCC15 were investigated by a Muse Cell Analyzer. DNA content index histograms of the control and treatment groups with crude extract and cisplatin in each cell cycle phase  are shown in After treatment with lupeol, citronellal and citronellal, G2/M enrichment significantly increased in SCC15 compared to the untreated control . This reTM Cell Analyzer following the Muse™ Annexin V & Dead Cell Kit procedure for the SCC15 cell line stained with annexin V and 7-AAD (7-amino-actinomycin D). The first and second quadrants represented dead cells and late apoptotic cells, respectively, while the third and fourth quadrants represented live cells and early apoptotic cells, respectively , while wound areas of treated cells with drugs (67.42±5.09%) and all extracts and compounds were widely open, with percentage range 46.45±5.64 to 63.01±2.42% (The wound closure assay is a method of 01±2.42% .2O2 (100 μM) was used as the positive control. Results showed that KL H, lupeol, citronellal and citronellol strongly induced ROS production in the SCC15 cell line. Interestingly, lupeol and citronellal stimulated high levels of ROS compared to the positive control (H2O2) in a dose-dependent manner. However, a low level of ROS was observed in the KL ET treatment. This finding indicated that high intracellular ROS generation was stimulated by crude extracts and their bioactive compounds  and active compounds  significantly increased the levels of pro-apoptotic, Bax and cleaved-caspase 3. Treatment of SCC15 with KL extracts and their compounds resulted in suppression of Bcl‑2 expression and decreased pro-caspase 3, showing similar results to the positive drug control, cisplatin . These fM1 macrophages express nitric oxide synthase (iNOS), TNF-α, IL-6, CD80 and CD86 costimulatory molecules. By contrast, M2 macrophages were defined based on the co-expression of CD163 (scavenger receptor) and CD206 (mannose receptor) as anti-inflammatory markers and arginase-1 (ARG-1) ,34. We nTM Cell Analyzer. Activated macrophages significantly induced cell apoptosis of SCC15 in the co-culture condition  polarized THP-1-derived macrophages toward M1. Next, we investigated the effect of crude extracts and compound-activated macrophages involved in anti-cancer activity. In this experiment, THP-1 cells were placed in the upper chamber and stimulated with PMA, followed by resting in fresh complete RPMI for 2 days. THP-1-derived macrophages were cultured with 20 μg/ml of lupeol, citronellal and citronellol and 100 μg/ml of crude hexane, and crude ethanol before being incubated for 24 hours and co-cultured with SCC15 at day 6, as shown in ondition . The relondition . CollectDioscorea nipponica (dioscin) in SCC15 [Rheum undulatum L. in HN22 and SCC15 [Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. The incidence of HNSCC continues to rise and is anticipated to increase by 30% by 2030 . Moreovein SCC15 , Rheum und SCC15  and cannnd SCC15 . In thisMoringa oleifera Lam., extract and compound 3-HBI inhibited cell proliferation and induced apoptosis in SCC15 cell line through the activation of cleaved caspase-3 and Bax as well as suppressing anti-apoptotic factor, Bcl-2 [et al. suggested that the cell cycle of K562 (chronic myelocytic leukemia) was significantly arrested at the G2/M phase after treatment with a subfraction of kaffir lime leaves for 24 hours [Our previous study explored r, Bcl-2 . Crude e24 hours . In this24 hours . Our curExcessive increase in intracellular ROS induced DNA damage, cell cycle arrest and senescence as well as apoptosis of tumor cells . Our stuin vitro techniques were used. In general, some cytokines activate M1 or M2 polarization in macrophages generated from in vitro culture. Nitric oxide synthase (iNOS), TNF-α, IL-6, and the costimulatory molecules CD80 and CD86 were expressed by M1 macrophages. In contrast, M2 macrophages were identified based on the expression of the anti-inflammatory markers arginase-1 and CD163 (scavenger receptor and mannose receptor) [Since immunotherapeutic strategies that target macrophages in the treatment of cancer, particularly HNSCC, are a potential approach. We thus evaluate the immunomodulation properties of kaffir lime leaf extracts and their constituents on inducing macrophage polarization against SCC15 in addition to the direct anti-cancer activity of these substances. Conventional classifications of macrophage polarization include naive macrophages, which rapidly differentiate into the other 2 main phenotypes of classically activated macrophages (M1) and alternatively activated macrophages (M2). In most investigations on macrophage polarization, straightforward eceptor) . Macropheceptor) . Due to eceptor) . M1 macreceptor) . Variouseceptor) ,13. Theret al. reported that phospholipase D4 (PLD4) were involved in the activation process of M1 phenotype macrophages and developed antitumor effects in colon cancer cells [et al. showed that cold plasma treatment stimulated the differentiation of pro-inflammatory (M1) macrophages to prevent solid cancer progression via release of anti-cancer cytokines [et al. concluded that Clinacanthus nutans extracts reduced pro-inflammatory condition in the co-culture of triple-negative breast cancer cells (MDA-MB-231) and THP-1 macrophages [In this study, we investigated whether polarization of macrophages toward M1 or M2 types might be induced by KL extracts and active compounds. Cells were cultured with kaffir lime leaf extracts and their constituents lupeol, citronellal, and citronellol in order to better understand the polarization phenotype of macrophages. The number of CD80 positive cells was significantly larger in macrophages treated with extracts and their components compared to the untreated control, according to flow cytometry used to identify M1 and M2 positive cell populations . The finer cells , while aer cells . Kaushikytokines , while Nrophages .The Nf-κB and the STAT pathways are known to play pivotal roles in the transcriptional profile of macrophages. Among the transcriptional factors, STAT1 and canonical Nf-κB (p50/p65 heterodimer) were essential for M1 tumoricidal functions and triggered the expression of pro-inflammatory cytokines . LPS is Our findings highlighted the potential of crude KL extracts and their compounds lupeol, citronellal and citronellol in stimulating M1 type polarization against cancer via TNF-α induced apoptosis. However, further studies could investigate whether KL and its bioactive compounds activate STAT1 and Nf-κB. It is suggested to use a variety of surface markers and confirm TAM activity by comparing it to a positive control in order to trigger apoptosis due to the limits of our work to apply biomarkers for the M1 and M2 phenotype. Different HNSCC cancer cell types must validate the extract’s direct action, and these cells must also test various doses and time periods. The use of patient-derived xenograft models and HNSCC patient-derived samples in future studies will be very effective in examining combination effects and overcoming resistance mechanisms.This study explored the anti-proliferative effects among KL extracts from various solvents as hexane and ethanolic extracts and their contained compounds including lupeol, citronellal and citronellol on the SCC15 cell line. This plant extract and compounds inhibited cell growth and triggered apoptosis through activation of the pro-apoptotic Bax and inhibition of the anti-apoptotic Bcl-2, thereby inducing the downstream caspase-dependent apoptosis pathway, caspase-3 in SCC15. Treatment with KL extracts and their contained compounds significantly increased cell cycle arrest at the G2/M phase of SCC15. They were also ROS inducers associated with high apoptosis and G2/M enrichment. Interestingly, this was the first report of the immunomodulatory effect of crude KL extracts and their contained compounds that polarized M1-macrophages and increased TNF-α production, resulting in apoptosis of SCC15 in co-culture with stimulated macrophages . Thus, bS1 Raw images(PDF)Click here for additional data file.


No current model of consciousness is univocally accepted on either theoretical or empirical grounds, and the need for a solid unifying framework is evident. Special attention has been given to the premise that self-organized criticality (SOC) is a fundamental property of neural system. SOC provides a competitive model to describe the physical mechanisms underlying spontaneous brain activity, and thus, critical dynamics were proposed as general gauges of information processing representing a strong candidate for a surrogate measure of consciousness. As SOC could be a neurodynamical framework, which may be able to bring together existing theories and experimental evidence, the purpose of this work was to provide a comprehensive overview of progress of research on SOC in association with consciousness.A comprehensive search of publications on consciousness and SOC published between 1998 and 2021 was conducted. The Web of Science database was searched, and annual number of publications and citations, type of articles, and applied methods were determined.n = 12 studies. EcoG data were assessed in n = 4 articles, fMRI in n = 4 studies, and EEG/MEG in n = 10 studies. Notably, different analytical tools were applied in the EEG/MEG studies to assess a surrogate measure of criticality such as the detrended fluctuation analysis, the pair correlation function, parameters from the neuronal avalanche analysis and the spectral exponent.A total of 71 publications were identified. The annual number of citations steadily increased over the years. Original articles comprised 50.7% and reviews/theoretical articles 43.6%. Sixteen studies reported on human data and in seven studies data were recorded in animals. Computational models were utilized in Recent studies pointed out agreements of critical dynamics with the current most influencing theories in the field of consciousness research, the global workspace theory and the integrated information theory. Thus, the framework of SOC as a neurodynamical parameter for consciousness seems promising. However, identified experimental work was small in numbers, and a heterogeneity of applied analytical tools as a surrogate measure of criticality was observable, which limits the generalizability of findings. Consciousness has been fascinated humankind since its very beginning and the relationship between the aware perception of stimuli and the orchestration of billions of neurons in the brain still is a baffling topic for many researchers all over the world . Over thTherefore, the purpose of this work was (i) to review the literature including annual number of publications and citations, type of articles, and applied methods, as well as to outline evidence bringing together theories and experimental evidence on how SOC may account for conscious perception and (ii) to give a conceptual perspective regarding agreements with the most influencing theories on consciousness described in the following.Theories on how consciousness relates to the physical domain generally start from different premises. Hence, the current two main theories of consciousness, the global workspace theory and the integrated information theory, incorporate distinct approaches to the definition of consciousness . The gloϕ, which reflects the amount of causally effective information that can be integrated across a subset of elements  uses a distinct approach claiming that the existence of consciousness cannot be inferred starting from physical systems . In theielements . Due to elements , the IITelements .Moreover, special attention has been given to the hypothesis that neural dynamics might be governed by the phenomenon of SOC. SOC, in the sense of statistical physics, is defined as a specific type of behavior, seen when a system undergoes a phase transition. During a phase transition, macroscopic properties of the system, termed the order parameters, change as a function of a so-called control parameter. For example, when water gets boiled, a phase transition from liquid to a vaporous phase occurs. Here, the order parameter would reflect the phase’s entropy (such as water or vapor), whereas the control parameter is the temperature. Modifying the control parameter gradually changes the order parameter until a specific point, at which the values of the order parameter vary abruptly. Graphically, phase transitions are either marked by a discontinuity of the phase diagram (a jump of the order parameter) or by a point of non-differentiability reflected as a sharp corner. The latter is termed a continuous second order phase transition, which allows the system to be poised exactly between two phases. In that case the system is in the critical state, residing between two qualitative distinct types of behavior such as ordered and disorder. A system at criticality is therefore sometimes referred to as on the “edge of chaos.” If the control parameter is below the critical value, the state is called subcritical, whereas values above the critical state result in a supercritical state . SystemsAlan Turing was probably the first one speculating that the brain could be in a critical regime in his seminal paper on the topic of artificial intelligence written in 1950 . A decadin vivo experiments confirmed power law statistic and spontaneous activity in form of neuronal avalanches in cats under anesthesia  of mature organotypic cultures and acute slices of rat cortex using a multielectrode array . Indeed,esthesia , in awakesthesia  and in resthesia . First sesthesia . In 2013esthesia . Using iesthesia . Subsequesthesia .σ, which is defined as the ratio of descendants, the number of events in a temporal interval t and the ancestors, the number of events in the following interval t + 1  were performed with the keywords (i) “self-organized criticality” OR “critical dynamics” OR “neuronal avalanche” AND “brain” as well as (ii) “consciousness” OR “conscious percept” AND “brain” OR “neural activity.” In addition, to quantify the growth in the field of SOC and consciousness with respect to the trends of the topics separately, the ratio between the number of citations was normalized by the total number of citations identified with the search (i) and (ii).Data were retrieved from the Web of Science (WoS) electronic database (SCI-Expanded). A search (All-Fields) with the keywords: “self-organized criticality,” OR “critical dynamics,” OR “phase transition,” OR “neuronal avalanches,” AND “consciousness” OR “conscious percept” was performed, Articles published between 01.01.1998 and 31.12.2021 were included. There were no language restrictions applied. The data were extracted in December 2021 from the Web of Science and exported to plain text. The data were then tabulated in a spreadsheet using Microsoft Excel . In total n = 71 publications were included. These had a total number of 1,968 citations , an h-index of 21, and 24.3 citations averaged per item. Most articles were original . This was followed by 31 reviews/theoretical articles (43.6%), three proceedings papers (4.2%) and one editorial (1.4%). The annual number of publications was highest in the year 2020 with a local maximum in 2017. The annual number of citations steadily increased over the years  reported on human data and in n = 7 (19.4%) studies data was recorded in animals. Regarding the methods, computational models were utilized in n = 12 studies (n = 4 articles (n = 4 studies (n = 4 studies (n = 10 studies (n = 3), the pair correlation function (n = 2), parameters from the neuronal avalanche analysis (n = 2), and the spectral exponent (n = 1), whereas one study applied multiscale entropy and permutation entropy and another one the autocorrelation function of global alpha oscillations.Out of the original articles,  studies , electroarticles , fMRI in studies , voltage studies , EEG/MEG studies  and EEG  studies . Notablyn = 473), followed by G. Werner from the University of Texas with 143 citations from In total, 252 authors were identified. The author with the highest number of publications was U. Lee affiliated with the University Michigan, United States, followed by E. Tagliazucchi from Netherlands Institute for Neuroscience. The latter also had the highest number of citations , the IIT had the highest increase in publication and citation count .To note, findings of critical dynamics in the brain did receive substantial critiques . For insΦ). Here, research on criticality and consciousness is largely informed by network models. For instance, Kim and Lee computed a large-scale human brain network model implementing a Kuramoto model on the scaffold of an anatomically informed human brain network structure constructed from diffusion tensor imaging  was determined as the minimum amount of information that is lost when splitting the system into two-subsystems introduced by Φ and metastability was observed. This findings led the authors to conclude that the two influential theories GWT and IIT could be compatible and that the criticality hypothesis offers a framework in which experimental predications from both can coexist (Whereas the global workspace theory and the IIT address distinct aspects of consciousness, the first conscious access closely related to the function of conscious awareness and the latter the phenomenology of consciousness, one notable study combined both in the context of criticality . Enzo Ta coexist .Thus, summarized, the formulation of a regime within the IIT in which segregation and integration occur simultaneously at its maximum corresponding to the conscious state goes hand in hand with the critical state from a dynamical perspective . FurtherThe main limitation of this study is that the analysis was solely based on Web of Science. Web of Science covers approximately 34,000 journals with over 75 million records and includes the sub-databases Science Citation Index, Social Sciences Citation Index, Arts and Humanities Citation Index, Conference Proceedings Citation Index, Book Citation Index, and Emerging Sources Citation Index . FurtherThe topic is compelling as principles such as SOC describing outcomes of collective phenomena in any complex dynamical system, provide a model suitable to situate the phenomenon of consciousness within universal laws of the physical world . WhereasThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.NW conceptualized the study, analyzed the data, and wrote the manuscript. TH supervised the project and revised the final version of the manuscript. All authors discussed the results, contributed to the article, and approved the submitted version.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.


Auf das südliche Afrika verteilte sich vor der Pandemie circa 2 % des weltweiten internationalen Reiseaufkommens. Davon erreichten fast die Hälfte dieser jährlichen Ankünfte Südafrika. Während der Pandemie hat sich gezeigt, dass gerade die Zugpferde des internationalen Tourismusmarketing der Region, die Safarilodges und Naturerlebnisanbieter, ohne Ankünfte aus Übersee nicht zu bewirtschaften sind. Als Alternative für diesen Nachfrageeinbruch wird einer Steigerung der Regional- und Binnennachfrage kaum Potenzial zugestanden. Dieser Standpunkt soll hier bestritten werden, ohne dabei jedoch vom Alleinstellungsmerkmal des Safaritourismus abzurücken. Die Empfehlung zielt stattdessen auf eine Erweiterung des Angebotsspektrums ab. Dabei werden 3 Absichten verfolgt: Erstens, den Mangel an der Binnennachfrage für die klassischen Reiseangebote der Region zu ergründen, zweitens Chancen für eine Aufwertung des Afrikabildes darzulegen und drittens Potenziale für eine Produktdiversifizierung aufzuzeigen, die auch eine wachsende Regional- und Binnennachfrage in den Blick nimmt. Big 5 kein Begriff sind, lernt ihn spätestens bei der Ankunft am Tor des Safariparks kennen. Dass dieser zum Markenkern des Safaritourismus gewachsene Sammelbegriff für Leopard, Büffel, Löwe, Elefant und Nashorn der kolonialen Trophäenjagd entstammt, erfahren Reisende selten. Dass es nun ab der ersten Safari darum geht, möglichst viele dieser Tiere zu erspähen, ist aber schnell als Erfolgsziel ausgemacht. Safaritourismus ist das Alleinstellungsmerkmal Ostafrikas und des südlichen Afrikas, dem geographischen Untersuchungsgegenstand dieses Artikels. Insbesondere charismatische Säugetiere gibt es in vergleichbarem Artenaufkommen in keinem anderen Zielgebiet der Welt zu fotografieren.Wem die Dennoch ist das südliche Afrika im globalen Vergleich nur sehr gering in internationale Reisemärkte eingebunden und auch regional ist die touristische Reisetätigkeit schwach. Mit Regionaltouristen sind hier Reisende gemeint, die sich zwischen den Staaten der Region bewegen.Studien zu Binnentourismus für die Region sind sehr begrenzt Rogerson . Viele LInternationale Tourismusankünfte auf dem Kontinent werden von globalen Statistiken in den Schatten gestellt. Gemessen an den letzten Daten der UNWTO vor dem Ausbruch der Pandemie, gab es 2019 weltweit 1,464 Mrd. internationale Touristenankünfte. Afrikas Anteil daran betrug 68,6 Mio. oder knapp 4,7 % UNWTO , S. 4. NFür die meisten Destinationen sind die Daten zu Touristenankünften in der Region jedoch höchst unzuverlässig, vor allem deshalb, weil vielerorts klandestine Arbeitsmigranten als Touristen erfasst werden. Dies betrifft nicht nur Einreise-, sondern auch Transitdaten. Überseereisende, die mit hoher Sicherheit größtenteils als Urlaubs- oder Geschäftstouristen gelten können und auch umfänglich touristische Dienstleistungen nutzen, machen deutlich kleinere Anteile unter den Ankünften aus: Diese Verteilung erstreckt sich unter den Staaten des südlichen Afrikas zwischen 12,7 % in Botsuana, einem Land mit sehr hohem Transitanteil und 28,1 % in Südafrika, dem beliebtesten Zielland für klandestine Arbeitsmigration in der Region . Letzteres betrifft größtenteils die urbane weiße Mittel- und Oberschicht Südafrikas. Doch gerade in Südafrika wäre die Ausschöpfung eines breiteren Nachfragepotenzials auch in gehobenen Marksegmenten möglich. Im Jahr 2019 wurden dort 54 % der oberen 10 % aller Einkommen von der ehemals unterdrückten schwarzen Mehrheitsbevölkerung erwirtschaftet . Viele Destinationen bieten den eigenen Bürgern zwar Rabatte auf die Parkgebühren an. Vergünstigte Tagesausflüge in die Parks sind in den meisten Destinationen üblich und haben seit Pandemiebeginn stark zugenommen. Doch es gibt weitere Hindernisse, die viele Einheimische von längeren Aufenthalten in den Lodges abhalten. Neokoloniale Untertöne können Einheimische abschrecken Florio . Gerade Big-5-Tourismus aber nur eine Randrolle. In der Regel wird einmal im Reiseablauf zwischen Safaripark und Lodge am Wegesrand kurz Halt gemacht, um in einem eigens für diesen Zweck errichteten Kulturdorf  ein paar Bilder von Einheimischen vor ihren Hütten zu machen. Leider findet hier jedoch wenig Vermittlung von Kulturgeschichte in Form von konkreten Inhalten und Sehenswürdigkeiten statt, obwohl diese existieren. Kultur wird meist auf ein austauschbares Vortanzen bzw. Vorsingen der entsprechenden Volksgruppe begrenzt, in dessen Heimat man sich gerade befindet. Was für manch einen Überseetouristen wohl als halbwegs authentische Darstellung der lokalen Kultur durchgehen mag, kann für Menschen, die selbst aus diesen oder verwandten Kulturgemeinschaften kommen, zumeist vor allem eines sein: peinlich.Seit über 40 Jahren befasst sich die Tourismuskritik mit ungleichen Machtverhältnissen zwischen Besuchern und Besuchten  als auch das Einbettungspotenzial von Narrativen in Destinationskulissen (Instragrammability). Letztere sind wichtige Faktoren für Reiseentscheidungen, gerade für afrikanische Quellmärkte, die fast ausschließlich aus jüngeren Generationen bestehen (Gumpo et al. Ein Bewusstsein für die Rolle der sozialen Medien für die Ausgestaltung von Marketingnarrativen im Tourismussektor ist zwar längst in der Region vorhanden. Abgesehen von Südafrika, das auch in diesem Bereich eine Vorreiterrolle auf dem gesamten Kontinent spielt, gibt es aber bei Destinationen, die sich auch jenseits der Südafrikas Marketingstrategie und -kampagne für den Inlandstourismus „Sho’t left“ (Rogerson und Lisa Erfolgreich sind vor allem Produkte, die kompatibel mit Gruppeninteressen, zeitlich begrenzt und nicht zu kostspielig sind. Zielgruppen können Familienverbünde sein, aber auch Freundesgruppen oder Liebespaare. Stattdessen werben viele Destinationen, beispielsweise die Viktoriafälle oder die Drakensberge, mit aufwendigen Abenteuerprodukten, die für regionale Märkte nicht nur deutlich zu viel Nervenkitzel ankündigen, sondern auch leere Brieftaschen.Reenactment kolonialer Rollenbilder bedient; und eine aufstrebende afrikanische Mittel- und Oberschicht, die neue Reisebedürfnisse hat, die mehr mit einer digitalisierten Moderne als mit der Ästhetik des ausgehenden 19. Jahrhundert zu tun hat.Letztlich haben wir es bei der vorgestellten Thematik im Wesentlichen mit 3 Trends zu tun: ein beispielloser Abbruch interkontinentaler Ankünfte in der Region über einen Zeitraum von anderthalb Jahren; ein etablierter Big-5-Tourismus, der sein Erscheinungsbild auf eine kleine aber vergleichsweise zahlungskräftige Zielgruppe ausrichtet und sich dabei kolonialer Designs bis hin zu einem Storytelling ausgestalten. Langfristig bedarf es einer Dekolonialisierung der Eigentumsverhältnisse touristischer Betriebe. Zur Aufwertung der Erlebnisprodukte ist aber dringend auch eine deutliche Aufwertung kultureller Reiseinhalte durch die Inhaber des kulturellen Eigentums selbst gefragt.Natürlich ist es schwierig bis unmöglich nach 2 langen verlustreichen Geschäftsjahren in neue Angebote und Ausstattungen zu investieren. Es ist leicht nachzuvollziehen, dass eine Angleichung von Übernachtungspreisen an regionale Einkommen auch zu Qualitätseinbußen im Angebot führen kann. Doch diese Einschätzungen sind buchstäblich zu kurz gedacht. Der Safarisektor ist gut beraten, an seinen Kernangeboten und Märkten festzuhalten. Zugleich sollte der Sektor sich konzeptionell öffnen, indem er zumindest beginnt, mit neuen Produktlinien zu experimentieren und eine inklusivere Zielgruppenstrategie zu fahren. Destinationen sollten kulturelle und historische Angebote nicht nur stärker ausbauen, sondern diese inhaltlich auch mit marktorientiertem Big 5 auch an Bigger Ideas.Sowohl das Afrikabild in Übersee als auch die Reisebereitschaft in den Binnen- und Regionalmärkten Afrikas befinden sich in einem Wandel, der nach der weltweiten Eindämmung der Pandemie und einer hoffentlich damit einsetzenden wirtschaftlichen Erholung in der Region, an Dynamik hinzuzugewinnen verspricht. Um auf diesen Zug aufzuspringen, bedarf es neben den


Euryarchaeota , abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM), such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island  to metagenome-assembled genomes to determine the diversity of transcriptionally active Ca. Poseidoniales. Summer metatranscriptomes had the highest abundance of Ca. Poseidoniales transcripts, mostly from the O1 and O3 genera within Ca. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly from Ca. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the ß-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific to Ca. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment of Ca. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundant Ca. Poseidoniales in nearshore and inshore waters. Together, our data suggest that Ca. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.Marine Group II  Euryarchaeota,2 definitive determination of their physiology and ecological roles has remained challenging due to the lack of a cultivated isolate. Nonetheless, as data describing MGII distributions throughout the ocean have increased, several patterns have emerged: MGII is often highly abundant in the euphotic zone and in coastal waters, can reach high abundance following phytoplankton blooms, and largely comprised two subclades, MGIIa and MGIIb.4 Early metagenomic studies provided the first evidence that MGII may be aerobic (photo)heterotrophs,7 a hypothesis supported by incubation experiments10 and by the gene content of diverse metagenome-assembled genomes (MAGs).15 Two recent studies deepened our understanding of the phylogenomics and metabolic potential of MGII by analyzing hundreds of MAGs, highlighting clade-specific differences in genomic potential for transport and degradation of organic molecules, light-harvesting proteorhodopsins, and motility.17 Here, we refer to MGII as the putative order “Candidatus Poseidoniales,” MGIIa and MGIIb as the putative families “Ca. Poseidoniaceae” and “Ca. Thalassarchaeaceae,” respectively, and putative genera as specified by Rinke et al.16 We occasionally use “MGIIa” and “MGIIb” for consistency with previous literature.Since the initial discovery of Marine Group II (MGII) Ca. Poseidoniales transcripts can be abundant in marine metatranscriptomes, suggesting transiently high transcriptional activity.19 When metatranscriptome reads from the Gulf of Aqaba were mapped to metagenomic contigs from the Mediterranean Sea, genes involved in amino acid transport, carbon metabolism, and cofactor synthesis were highly transcribed in the aggregate euryarcheal community.21 In another study, mapping deep-sea metatranscriptome reads to novel Ca. Poseidoniales MAGs indicated transcription of genes related to protein, fatty acid, and carbohydrate transport and metabolism, likely fueling aerobic heterotrophy.15 Finally, a metaproteomics study found abundant euryarcheal transport proteins for L-amino acids, branched-chain amino acids, and peptides throughout the Atlantic Ocean.22 Despite these advances, little is known about the similarities or differences in gene transcription between Ca. Poseidoniaceae and Thalassarchaeaceae.Metatranscriptomics is one strategy for gleaning information about microbial activity in the environment. Ca. Poseidoniales in coastal waters near Sapelo Island . Prior work suggested that Ca. Poseidoniales are sporadically active at Sapelo Island23 and may comprise the majority of archea in mid-shelf surface waters of the South Atlantic Bight (SAB).24 Since other studies thoroughly described the genomic content of Ca. Poseidoniales MAGs, our focus instead was determining which of the clades were transcriptionally active and identifying highly or differentially transcribed genes. We used two Sapelo Island MAGs25 combined with recent Ca. Poseidoniales MAG collections17 to competitively recruit reads from a metatranscriptomic time series26 and an incubation experiment23 to determine which of the clades were active over time. We then used representative MAGs from highly active genera to determine which Ca. Poseidoniales genes were transcribed. Finally, we used quantitative PCR (qPCR) to measure the abundance of Ca. Poseidoniales 16S rRNA genes in DNA samples throughout the SAB to assess the prevalence Ca. Poseidoniales in this region.We report MAG-resolved metatranscriptomic analyses of 25) to previously reported Ca. Poseidoniales MAGs, including those binned from TARA Oceans, Mediterranean Sea, Red Sea, Gulf of Mexico, Guaymas Basin, and Puget Sound metagenomes32 . All genomes were converted to contig databases, and proteins were identified using HMMER,36 concatenated, aligned using MUSCLE,37 and used to build a phylogenomic tree using FastTree38 within anvi’o.The phylogenomic analysis compared SIMO Bins 19-2 and 31-1  as previously described.26 Briefly, 3–8 L of surface water was pumped through a 3-µm pore-size prefilter followed by a 0.2-µm pore-size collection filter, which was frozen on liquid nitrogen. RNA was extracted from the collection filter as previously described,26 including the addition of internal RNA standards to calculate volumetric transcript abundances.39 Analyses of “field” communities included Gifford et al. metatranscriptomes (iMicrobe Accession CAM_P_0000917)26 and the T0 metatranscriptomes from Vorobev et al.,23 while “dark incubation” analyses included only Vorobev et al. samples . Temperature, salinity, dissolved oxygen, pH, and turbidity data corresponding to metatranscriptome sampling times were downloaded from the NOAA National Estuarine Research Reserve System website .We used competitive read mapping to determine which 40) with the “very-sensitive” flag. Samtools v.1.3.1 (ref. 41) was used to index BAM files, which were profiled and summarized in anvi’o. Contig genus identity was imported to the anvi’o contig database as an external collection. The number of transcripts L–1 was calculated by scaling the number of mapped reads by the volume of water filtered and the recovery of internal standards (reported in refs. 26) as previously described.39 Seasonal transcript abundances were compared using a one-way ANOVA test in R42 with data log-transformed to improve normality. Significant groupings were defined post hoc with Tukey’s Honest Significant Difference (HSD) test using the agricolae R package.43Contigs from all MAGs used for phylogenomics  with 999 permutations.Nonmetric multidimensional scaling (NMDS) analysis of metatranscriptome hits was conducted using the vegan R package.VMDE00000000; SIMO Bin 31-1, VMBU0000000025) and one previously binned from Red Sea metagenomes . These MAGs represented genera O1, O3, and M, respectively, which were highly abundant in metatranscriptomes  using DIAMOND47 to search against all orthologous groups in eggNOG-mapper v.1 (refs. 46) and using the BlastKOALA portal .48 Putative genes for carbohydrate-active enzymes, peptidases, and membrane transport proteins were identified using HMMER searches of dbCAN2 (HMMdb v.7),50 MEROPS v.12.0 (ref. 51), and the Transporter Classification Database,52 respectively.MAGs were annotated using the archaeal database in Prokka v.1.13  by dividing by the total number of reads per sample. For each MAG, “highly transcribed” genes were 5% of putative genes with the highest median CPM across metatranscriptomes .Transcript reads were mapped to MAGs (combined into a single database such that each read mapped to only one MAG) to identify 53) was used to identify genes from each MAG that were differentially transcribed when each genus was highly transcriptionally active. For each MAG, “treatment” samples in DESeq2 were those where the respective genus recruited ≥50% of Ca. Poseidoniales reads from the metatranscriptome. Thus, positive fold-change values are genes transcribed at higher levels when the genus is highly transcriptionally active (compared to other metatranscriptomes). DESeq2 was also used to identify differentially transcribed genes for each MAG between T0 and T24 samples in high-tide (HT) dark incubations.23 Since T24 samples were the “treatment” condition in DESeq2, positive fold-change values here are genes transcribed at higher levels in T24 compared to T0 samples. In all DESeq2 analyses, genes with Benjamini–Hochberg-adjusted p < 0.1 were counted as having significantly different transcription.DESeq2 v.3.11  and genera O1 (SIMO Bin 19-2) and O3 : most summer samples had >1010Ca. Poseidoniales transcripts L–1, more than other seasons , with most mapping to O1  genes were involved in translation, transcription, replication/repair, or post-translation protein modification  MAG were not highly transcribed by Ca. Thalassarchaeaceae, including genes encoding a carbamoyl phosphate synthetase subunit (carA), a family 2 glycosyltransferase, chromosomal protein MC1b, and a ftsX-like permease. The carA gene had the highest median coverage of Ca. Poseidoniaceae genes across coastal metatranscriptomes , and proteorhodopsin . Differentially transcribed genes mapping to the O3 MAG were mostly depleted in O3-active metatranscriptomes and largely encoded proteins of unknown function; only the gene encoding ribosomal protein L12 was enriched in O3-active samples , while hits from the corresponding T24 samples were 98.1–99.3% Ca. Thalassarchaeaceae due to increased transcript hits to genus O1  but changed to 78.3–98.8% hits to Ca. Thalassarchaeaceae at T24 due to an increase in hits to O1  and a ligand-binding receptor for a general amino acid transporter  and multiple subunits of pyruvate dehydrogenase . None of the genes mapping to the O3 or M MAGs were transcribed at significantly different levels between HT incubation time points , with a range from 1.6 ×  104 to 7.6 × 108 genes L–1  and the mean efficiency was 93.4%  is among the highest measured in the ocean. Greater gene abundance in inshore, nearshore, and mid-shelf waters indicates that Ca. Poseidoniales are more abundant over the shallow shelf than further offshore , little is known about which clades are transcriptionally active in these regions. At Sapelo Island, the striking dominance of the Ca. Thalassarchaeaceae (MGIIb) genera O1 and O3 in summer samples, which also contained the highest levels of aggregate Ca. Poseidoniales transcripts  transcripts . A previous study found relatively high salinity and DOM enriched in marine-origin molecules over the mid-shelf SAB during July 2014.70 Our data suggest that Ca. Poseidoniaceae were relatively active over the shelf during this time and were transported inshore during flood tides, leading to shifts in transcriptional diversity between LT waters  and HT waters (which included a higher number of Ca. Poseidoniaceae).While numerous studies have demonstrated a high abundance of Ca. Poseidoniaceae (MGIIa) are the predominant euryarcheal family in many coastal ecosystems, particularly in summer (see refs. 72), but it is unclear what general patterns govern Ca. Poseidoniales distributions in coastal waters worldwide. Studies of Ca. Poseidoniales ecology often focus on distributions with depth, typically finding abundant Ca. Thalassarchaeaceae (MGIIb) in deeper waters and Ca. Poseidoniaceae (MGIIa) more prevalent in euphotic waters (see refs. 75). A recent mapping of global ocean metagenome reads showed that coastal populations of Ca. Poseidoniales were primarily Ca. Poseidoniaceae, though Ca. Thalassarchaeaceae MAGs recruited a substantial number of reads from some coastal metagenomes.17 Our data match this latter pattern, with Ca. Poseidoniales populations in surface waters off Sapelo Island dominated by highly active Ca. Thalassarchaeaceae  may explain the unusual pattern found in SAB waters: these genera peaked in summer at Sapelo Island, when water temperatures were 29–30 °C .We determined sets of “highly transcribed” genes mapping to MAGs of transcriptionally active Ca. Poseidoniales MAGs that were differentially transcribed when its genus was highly active  and our data indicate coastal Ca. Poseidoniales conform to this pattern, consistent with recent evidence from other regions.81 High transcription of proteorhodopsin supports the photoheterotrophic lifestyle hypothesized for Ca. Poseidoniales (see refs. 17).Our analysis revealed that es Figs.  and 3. PCa. Thalassarchaeaceae proteorhodopsin transcription. However, depletion of O1 crtD  transcripts during dark incubation  and the function of constitutive transcription is not straightforward: while some bacteria use proteorhodopsin to produce ATP when carbon-limited,84 high amounts of proteorhodopsin in other bacteria can physically stabilize membranes even when inactive.85 The high proteorhodopsin transcription in our data emphasizes, but provides little mechanistic clarification of, the physiological role for proteorhodopsin in Ca. Poseidoniales  putatively encodes a membrane-bound pyrophosphatase, which generates a proton or sodium ion gradient via hydrolysis of pyrophosphate, a by-product of numerous cellular processes.86 In metatranscriptomes from a phytoplankton bloom, enrichment of hppA transcripts suggested high pyrophosphate-based energy conservation in oligotrophic waters,87 and hppA transcripts were similarly abundant at night in an oligotrophic lake.88 Although widespread in MAGs from Ca. Poseidoniales,16hppA has not been previously recognized as a potentially important part of their metabolism. Our data suggest that Ca. Poseidoniales may be capable of using pyrophosphatase  to generate a protonmotive force  during incubations  may be adapted to clearer waters and reduce transcription in dark conditions.Although the Tons Fig.  suggestsCa. Poseidoniales may have been metabolizing proteins and fatty acids. High transcription of genes encoding proteases or peptidases from all Ca. Poseidoniales MAGs , active protein metabolism is consistent with previous experiments demonstrating protein assimilation10 and high transcription of Ca. Poseidoniales peptidase genes in other marine regions.21 In addition to genes encoding protein catabolism, high transcription of the 3-hydroxyacyl-CoA dehydrogenase gene from all three MAGs  but not Ca. Poseidoniaceae (MGIIa)17 and were among the most highly transcribed Ca. Thalassarchaeaceae genes  during incubations  with the high numbers of O1 pbuG transcripts in O1-active samples and dark-incubation endpoints  in Ca. Thalassarchaeaceae metabolism. In some phytoplanktons, pbuG is transcribed during nitrogen-stressed growth,96 potentially allowing access to DON. However, pbuG and xanthine dehydrogenase (xdh) are also transcribed when xanthine is catabolized by marine bacteria.97 Both Ca. Thalassarchaeaceae MAGs contain putative xanthine dehydrogenase genes : while all three MAGs contained this gene . They indicate an important role for Ca. Thalassarchaeaceae (MGIIb) as coastal photoheterotrophs, particularly in warm waters. High transcription of proteorhodopsin and membrane-bound pyrophosphatase genes suggested common methods for establishing proton gradients. Furthermore, high transcription of genes involved in protein/peptide metabolism and ß-oxidation of fatty acids confirmed peptide and lipid metabolism as a common trait. However, high transcription of Ca. Thalassarchaeaceae genes encoding amino acid-binding proteins and nucleotide transporters suggests uptake of these substrates may distinguish the two families. These data confirm the importance of DOM metabolism by Ca. Poseidoniales and suggest a potential role for organic nitrogen in Ca. Thalassarchaeaceae metabolism.Our metatranscriptomic data and associated experiments provide a novel window into the activity of SUPPLEMENTARY FIGURESSupplementary informationSupplementary informationSupplementary informationSupplementary informationSupplementary informationSupplementary informationSupplementary information


Tumor treating fields (TTFields) is an FDA‐approved adjuvant therapy for glioblastoma. The distribution of an applied electric field has been shown to be governed by distinct tissue structures and electrical conductivity. Of all the tissues the skull plays a significant role in modifying the distribution of the electric field due to its large impedance. In this study, we studied how remodeling of the skull would affect the therapeutic outcome of TTFields, using a computational approach.Head models were created from the head template ICBM152 and five realistic head models. The electric field distribution was simulated using the default TTFields array layout. To study the impact of the skull on the electric field, we compared three cases, namely, intact skull, defective skull, and insulating process, wherein a thin electrical insulating layer was added between the transducer and the hydrogel. The electric field strength and heating power were calculated using the FEM (finite element method).Removing the skull flap increased the average field strength at the tumor site, without increasing the field strength of “brain”. The ATVs of the supratentorial tumors were enhanced significantly. Meanwhile, the heating power of the gels increased, especially those overlapping the skull defect site. Insulation lightly decreased the electric field strength and significantly decreased the heating power in deep tumor models.Our simulation results showed that a skull defect was beneficial for superficial tumors but had an adverse effect on deep tumors. Skull removal should be considered as an optional approach in future TTFields therapy to enhance its efficacy. An insulation process could be used as a joint option to reduce the thermogenic effect of skull defect. If excessive increase in heating power is observed in certain patients, insulating material could be used to mitigate overheating without sacrificing the therapeutic effect of TTFields. Of all the tissues the skull plays a significant role in modifying the distribution of the electric field due to its large impedance. In this study, influence of remodeling of the skull on the therapeutic outcome of TTFields have been studied. This may provide new insights into the application scope of TTFields, and the strategy of utilizing skull defects to maintain field strength, avoid overheating, and maximize antineoplastic efficiency.22.1http://nist.mni.mcgill.ca/.Each patient's images were acquired through the local institutional review board review committee and informed consent was obtained from the patients. Patients with a new pathologic diagnosis of glioblastoma were included. Moreover, images were reviewed to ensure the selected tumors were in a single lobe or brainstem and harboring a significant central necrotic area. Necrotic areas, as the tumor cores, are necessary for analysis in FEM. Cranial MRI was acquired with the same 3T scanner. Radiological features were extracted from axial T1‐W, T2‐W and enhanced T2‐W images. The standard brain template ICBM152 image  was downloaded from the website 2.2https://itis.swiss/virtual‐population/tissue‐properties/database/dielectric‐properties/ Table The head template, ICBM152, and five realistic head models  created from MRI data were used to construct five head models with embedded tumors. After aligning the images to MNI  space, the coordinates of real head models were unified with the ICBM152.2.3The results generated by segmentation were all exported as NIFTI files. Then each tumor and the ICBM152 tissues were imported into the 3D slicer software and the “subtract” operation was run to solve tissue overlap issues. After five operations, the tumors were implanted into the template, that is, five head models with tumors were obtained.2.4Based on the location of the tumor, the position and size of the skull defect were determined by a senior neurosurgeon. Considering that the defective part of the skull would be filled with scalp tissue, the electrical properties of this part of the tissue were consistent with the scalp in the calculations of the skull defect models. Finally, the interpolation function of the tissue coordinates and the electrical properties of a head with a skull defect was established and assigned to the head model, that is, head models with a skull defect were obtained.2.5Usually, two pairs of arrays are attached to the patient's head, i.e., left–right arrays (LR arrays) and anterior–posterior arrays (AP arrays). In this study, to compare the effect of the skull defect on the electric field, only one pair of electric arrays was added, depending on the location of the skull defect. Each array consisted of a total of 3 × 3 transducers, which were electrode cylinders with a height of 1.0 mm and a radius of 10 mm. Based on the default layout of the electrode arrays (symmetric configuration) and the relative position of the transducers in the electrode arrays, the electrode sheets were added to the previously established head geometry model.2.6σ (S/m) is the electrical conductivity of tissues, ω is the angular frequency, ε is the relative permittivity, and E (V/m) is the electric field strength.Q (W/m3) is the heating power, J (A/m2) is the current density, and E* is the conjugate of electric field strength.The electric field distribution within the brain was calculated using the FEM to solve the electro quasistatic approximation of the Maxwell equations, which are valid for these models.2.7Considering the strong electric field strength at the defect site, there would be a significant increase in thermogenesis. To control the thermogenic effect, a scheme in this study, adding a thin electric insulating layer to the contact surface between the transducer and the hydrogel, was proposed. The electric insulating layer, with a thickness of 1 μm, a dielectric constant of 20 mm, a relative permittivity of 2.2, negligible conductivity, a specific heat capacity of 2.3 J/(kg·°C) and a heat conductivity of 0.42 W/m·K, was deposited on the inner surface of transducers at the site of the skull defect. Since the thickness of the insulating layer was negligible compared to that of the transducer, the boundary condition of contact impedance was applied to simplify the calculation.2.8The distribution of the intracranial electric field and the heating power of the gel were calculated according to above formulas, and were then exported and processed as colorful diagrams with color scales. Other data, including the average electric field strength and above‐threshold volumes (ATVs) of each tissue, were calculated in the COMSOL software and exported as needed.33.13. The ratio of males to females was 3:2.Patients with GBM and their imaging data were used in this study. The five included patients were 18–63 years old; their tumors were confined to the unilateral frontal, insular, temporal, and parietal lobes, and the brainstem, respectively; and the tumor volumes ranged from 22.36 to 114.28 cm3.2Depending on the tumor location, the electric field distribution was calculated only for a single pair of arrays, i.e., LR or AP arrays. Because the location of the skull defect and transducers were fixed, the skull defect only affected the pair of arrays with which there was a contact. The intracranial field distribution in the horizontal plane of each tumor is shown in Figure In addition, a “hot spot” formed at the interface of high and low conductivity tissues, which were not single points but regions with local field enhancement, as the field strength increased in the low conductivity tissues and decreased in the high conductivity tissues. In comparison, after bone flap removal, all models' tumor shell showed the expansion of volume and scope of “hot spot” activity except the brainstem model. Moreover, there was no significant decrease in hot spot scope and field strength after adding the insulating layer.In addition, at the tumor site, our region of interest, the field strengths of the tumor core, tumor shell and “brain”  were calculated, and the results are shown in Figures Comparing each case in the same model, Figure 3.3Although some encouraging results can be seen, we can increase the field strength to the tumor shell, as well as the intracranial field strength to some extent, by removing the bone flap. However, this created another problem i.e., the skin dermatitis, which is the main side effect of the electric field treatment, was caused by overheating of the electrode sheet and gel. While removing part of the skull increased the overall field strength due to the lack of the voltage dividing effect of the skull, the heating power of the gel increased. Concurrently, the cost of the temperature increase was reduction of voltage to maintain an equilibrium between heat production and dissipation, with the direct consequence that the increase in field strength was not sustainable, and the final intracranial field strength at equilibrium needed to be further inferred and calculated based on the heating power.Comparing the different models, it was obvious that the LR arrays had significantly larger heating power than the AP arrays, but the gap decreased after bone flap removal. What was found was that the increase in heating power was especially obvious after removing the bone flap in the insular lobe model, and the effect of the insulating layer on the heating power was different in each model.4In this study, we used a standard brain template, ICBM152, and intracranial lesions based on realistic head models derived from MRI data, to study the effect of skull defects on the intracranial electric field distribution and heating power in TTFields. We found that an increase in the intracranial electric field strength was accompanied by an increase in the heating power of gel in the presence of an incomplete skull. Skin dermatitis, the most common side effect of electric field therapy, is strongly correlated with heating power.Because there is no unified transducer array layout according to tumor location, size, and head parameters, and the object of this study was to investigate the effect of skull defects on the intracranial electric field distribution, the default array layout was adopted in each model in the study, and the calculation results only present the AP or the LR arrays, the pair of arrays that overlap with the location of the skull defect, since the effect of the skull defect on the other pair of arrays was almost negligible.Several in vitro experiments have shown that the lower threshold of 1.0 V/cm is the minimum magnitude for cell division and thus inhibition of cell line proliferation. Moreover, the effect is positively correlated with electric field strength, with the inhibition effect reaching a maximum at 2.5 V/cm.Since the therapeutic efficacy of electric field therapy is dose‐dependent and the average field strength of the tumor shell reached 1 V/cm when the skull was intact, the ATV 1.0 (to reach the minimum treatment threshold) and ATV 2.5 (to reach the maximum treatment effect) had greater significance. For all models, an increase in ATV 1.0 was observed when skull defects, especially in the frontal lobe model (44.71%) and the brainstem (17.6%), showed a significant increase in ATV 1.0. In addition, almost all tumor shells reached 1 V/cm after removal of the bone flap. Therefore, the elevated ATV 1.0 in the frontal lobe and brainstem may be related to their lower basal ATV 1.0. In other words, the field strength of the AP arrays was smaller than that of the LR arrays and removing the bone flap may enhance the therapeutic effect of the AP arrays to some extent. Moreover, all the models other than the brainstem model showed an extremely significant increase in ATV 2.5. These results suggest that removing the bone flap can improve the field strength of TTFields during treatment, especially for superficial tumors in the frontal and temporal lobes. For the insular lobe and brainstem models, several previous studies have demonstrated that although specific arrays can improve the electric field strength in the tumor shell, the results are still not ideal compared to superficial tumors, and removing bone flap can also improve the electric field strength in the tumor shell.Overall, Figure Enormalized denotes the normalized electric field strength, E2 indicates the average field strength in the skull defect or insulation process case, W1 is the maximum heating power for the skull intact case, and W2 is the maximum heating power for the skull defect or insulating process case.It is known that in practical clinical applications, the temperature of the transducer is monitored, and the voltage will decrease when it reaches 41°C. During operation, the device can lower the voltage to make the heating power equal to the heat dissipation power, so that the temperature is controlled near 41°C. Since our results were based on the calculation of the default voltage, the actual tumor shell field strength needed to be further calculated based on the heating power. Therefore, we introduced a definition of “normalized electric field strength”, using the heating power of the intact skull case as a benchmark for the treatment unification of each case. In other words, it may represent the actual change in the electric field strength when the transducer with the highest heat power was adjusted to that value in the case of skull defect or insulating process.In conclusion, Table This study is a computational modeling study and the physical parameters of each tissue have a large impact on the accuracy of the study; every effort has been made to avoid this error by selecting parameters provided by authoritative organizations. Field correlation and anisotropic conductivity, which have an effect on the field distribution and efficacy, are not included in this study.Taian Jin and Zhangqi Dou were responsible for the study methods, data analysis, writing, and editing. Yu Zhao was responsible for the analysis. Boxing Wei, Biao Jiang, Jinghong Xu, Buyi Zhang, and Fei Dong were responsible for the clinical concepts and data acquisition. Chongran Sun and Jianmin Zhang were responsible for conceptualization, guidance, and supervision.None.The Second Affiliated Hospital, Zhejiang University School of Medicine, Center Institutional Review Board approved this modeling study using the de‐identified publicly available data. All patients provided informed consent.