Background: Pyroptosis, a newly discovered type of programmed cell death, has both anti-tumor and tumor-promoting effects on carcinogenesis. In hepatocellular carcinoma (HCC), however, the associations between pyroptosis-regulated genes and prognosis, immune microenvironment, and immunotherapy response remain unclear.Samples and methods: Sequencing data were collected from The Cancer Genome Atlas database, The International Cancer Genome Consortium (ICGC), and The Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB). First, we investigated the expression levels and copy number variations (CNVs) of 56 pyroptosis genes in HCC and pan-cancer. Next, we identified 614 genes related to 56 pyroptosis-associated genes at the expression, mutation, and CNVs levels. Pathway enrichment analysis of 614 genes in the Hallmark, KEGG, and Reactome databases yielded a total of 253 significant signaling pathways. The pyroptosis-regulated genes (PRGs) comprised 108 genes that were derived from the top 20 signaling pathways, of which 57 genes had prognostic value in HCC. The least absolute shrinkage and selection operator (LASSO) analysis was performed to screen for PRGs with prognostic values. Ultimately, we constructed a risk score model with seven PRGs to predict HCC prognosis and validated its predictive value in three independent HCC cohorts. Risk scores were used to illustrate receiver operating characteristic (ROC) curves predicting 1, 3, and 5-years overall survival (OS). Single-sample gene set enrichment analysis (ssGSEA), was performed to study 28 types of immune cells infiltrated in HCC. The relationship between the risk signature and six immune checkpoint genes and immunotherapy was analyzed.Results: A total of seven PRGs were obtained following multiple screening steps. The risk score model containing seven PRGs was found to correlate significantly with the HCC prognosis of the training group. In addition, we validated the risk score model in two additional HCC cohorts. The risk score significantly correlated with infiltrating immune cells , ICB key molecules , and ICB response.Conclusions: This study demonstrated a vital role of PRGs in predicting the prognosis and immunotherapy response of HCC patients. The risk model could pave the way for drugs targeting pyroptosis and immune checkpoints in HCC. Liver cancer was ranked sixth in cancer incidence and fourth in tumor-related mortality globally . Each yePyroptosis, a form of regulated cell death, is triggered by inflammatory caspases and characterized by the cleavage of pore-forming effector proteins: gasdermins . In the Pyroptosis is becoming a popular topic as it is involved in all stages of tumor progression. In stomach cancer, decreased GSDMD expression enhanced tumor growth by accelerating cell proliferation . AnotherAs previously stated, accumulating evidence suggests that pyroptosis plays an important role in predicting prognosis and regulating the immune environment across multiple cancers. Based on a risk model consist of 15 pyroptosis-related lncRNAs, researchers found that the high-risk group displayed higher immune infiltration fraction and activity in glioblastoma . In a pyhttps://xenabrowser.net/datapages/). The “CGDSR” R package was used to obtain corresponding clinical information. We used the “TCGA biolinks” R package to download the mutation data. The training group comprised 370 HCC patients with survival data from the TCGA-LIHC database. For model validation group 1, we obtained 243 HCC patients’ expression matrix and clinical information from the ICGC database (https://dcc.icgc.org/releases/current/Projects). Validation group 2 consist of two cohorts of HCC patients with expression and survival data, HCCDB6 (n = 209)and HCCDB17 (n = 94), which were obtained from the HCCDB database (http://lifeome.net/database/hccdb/home.html). All survival data and clinical parameters from the training and validation groups are summarized in Supplementary Table S1_TCGA_clin.xlsx, Supplementary Table 1_ICGC_clin.xlsx, and Supplementary Table 1_B6_17_clin, respectively. https://www.gsea-msigdb.org/gsea/msigdb/) (58 pyroptosis genes were obtained from the literature  (PMID338msigdb/) . The TCGThe expression patterns, genetic variations, and survival analysis of pyroptosis genes in various cancer typesThe mRNA levels of 56 pyroptosis genes were compared using the Wilcoxon test between normal liver tissue and HCC samples. The expression profiles of 56 pyroptosis genes in ESCA, LUSC, LUAD, STAD, COAD, and READ were determined. The copy number variations (CNVs) of pyroptosis genes were also examined in pan-cancer. The log-rank test was used to compare the differences in overall survival (OS) between the high and low groups based on pyroptosis gene expression.p-value of <0.05. Secondly, we carried out pathway enrichment analysis to find out which pathways are the 614 genes involved in. Using the tools of KEGG (https://metascape.org/)  , we screThe “ClusterProfiler” R packages were used to perform functional enrichment analysis of candidate genes . The TopSurvival analysis revealed that 57 out of the 108 key genes have prognostic value in HCC. The 57 key genes were subjected to Lasso regression analysis  and a rigenei*coefi).Risk score = ∑ is an extension of the GSEA method, which allows the definition of an enrichment score that represents the absolute degree of enrichment of the gene set in each sample within a given dataset . To deteThe relationship between six immune checkpoints  and risk group were examined.https://tcia.at/home) (Immunophenoscore (IPS) could act as a good predictor of CTLA-4 and PD-1 responsiveness as well as the response to immunotherapy. The immune checkpoint inhibitor (ICI) immunophenoscore file was obtained from the Cancer Immunome Database  . We thenp values are two-side and p < 0·05 represents statistical significance. In the statistical graph, ns denotes p ≥ 0.05, * denotes p < 0.05, **: denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001.ROC curves and the area under curves (AUC) were computed using the “pROC” and the “timeROC” packages, respectively. Analyses between the two or more groups were performed using the Wilcox test and the Kruskal Wallis test, respectively. We used the Kaplan-Meier method for survival analysis and the log-rank test to calculate the significance of differences. All statistical analyses were performed in R 4.3.0 and SPSS16 software. All statistical In comparison to normal controls, 46 out of the 56 (82.1%) pyroptosis genes were dysregulated in HCC. Thirty-six pyroptosis genes exhibited significantly higher transcriptional levels in HCC, including APIP, BAK1, BAX, CASP3, CASP4, CASP6, CASP8, CASP9, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHMP7, CYCS, DFNA5, DHX9, GPX4, GSDMB, GSDMC, GSDMD, HMGB1, IL1A, IRF2, NLBP1, NLRP9, NOD1, NOD2, PLCG1, PRKACA, PYCARD, SCAF11, TIRAP, and TP53. In contrast, the expression levels of ten pyroptosis genes, including AIM2, ELANE, IL-1β, IL6, NLRC4, NLRP3, NLRP6, NLRP7, TNF, and TP63, were significantly lower than those of healthy controls . In subgSubsequently, we examine the expression levels of 56 pyroptosis genes between pan-cancer and their corresponding normal controls, including esophageal squamous carcinoma (ESCA), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ). The majority of pyroptosis genes exhibited significant dysregulation in tumor tissues compared to normal controls .Meanwhile, we explored the copy number variations (CNVs) of pyroptosis genes in pan-cancer. The result showed the 56 pyroptosis genes had multiple CNVs in these cancers. Here, we used the CNVs modification of four genes  in pan-cancer as an example .Tumor samples were divided into high and low expression groups based on the median value of gene expression. According to the results of the survival analysis, 14 out of the 56 Pyroptosis genes were significantly associated with HCC prognosis. The high expression of BAK1, BAX, CASP4, CHMP2B, CHMP3, CHMP4A, CHMP4B, DFNA5, DHX9, IL-1A, IRF2, NLRP6, NOD1, and SCAF11 predicted poor prognosis in HCC .p-value (padj) < 0.05, we obtained 9,759, 18,063, and 7,509 pyroptosis-related genes at the expression level, mutation level, and CNV level, respectively. Using the intersection set, 614 candidate genes associated with pyroptosis genes at all three levels were obtained. To comprehensively study the genes regulated by the pyroptosis gene, correlation analysis was performed between the 56 pyroptosis genes and the remaining genes at the expression, mutation, and CNV levels. By setting a threshold of |correlation coefficient| (| Cor |) > 0.5 and adjusted p < 0.05, Supplementary Table 5_corsigs_GOBP.xlsx, Supplementary Table 5_corsigs_GOCC.xlsx, Supplementary Table 5_corsigs_GOMF.xlsx).We used the “ClusterProfiler” R packages for functional enrichment analysis of 614 candidate genes. The result showed that these candidate genes were strongly associated with nuclear-transcribed mRNA catabolic process, translational initiation, and other biological processes, suggesting were involved in gene expression and protein synthesis. The top10 entries for biological process (BP), molecular function (MF), and cellular component (CC) are shown . Using the metascape database, we performed pathway enrichment analysis against the Hallmark, KEGG, and Reactome pathways to investigate the signaling pathways in which 614 candidate genes are involved. 253 pathways were identified using a We designated 108 genes that participated in the pyroptosis-regulated pathways as pyroptosis-regulated genes (PRGs) (Supplementary Table 7_sig_gene. xlsx). The CNVs, mutations, and expression of 48 PRGs in the key pathway “Genes from Metabolism of RNA” were systematically studied. The results showed that 48 PRGs exhibited varying degrees of CNV alterations and mutation levels in pan-cancer . To exclude the overfitting, we performed the LASSO-Cox regression on 57 PRGs and identified a total of seven PRGs in the training group. The screening process and results are displayed in To determine the power of the risk score model in predicting HCC prognosis, we separated training group patients into high- and low-risk groups according to the best cut-off value. The risk scores distribution of the two groups is shown in To further establish the predictive power of the risk score model, we validated it in an independent dataset ICGC. Patients were divided into the low-risk and high-risk groups based on the optimal cut-off value. In the training cohort, we analyzed the relationship between clinical manifestations and the risk score. + T cells, activated dendritic cells, central memory CD4+ T cells, effector memory CD4+ T cells, immature dendritic cells, regulatory T cells, type 2 T helper cells compared with the low-risk group. Meanwhile, high-risk group displayed lower infiltration levels of eosinophils and memory B cells . Pearson correlation analysis was performed to assess the association between risk score and the abundance of immune cells. As shown in + T cells, activated dendritic cells, central memory CD4+ T cells, effector memory CD4+ T cells, MDSC, regulatory T cells, and type 2 T helper cells and negatively correlated with CD56bright natural killer cells, effector memory CD8+ T cells, eosinophils, mast cells, memory B cells, nature killer cells, neutrophils, and type 1 T helper cells.Next, we determined the pattern of immune cell infiltration in 370 HCC patients using the ssGSEA . Resultsp < 0.0001) and CTLA‐4 (p < 0.001) expression was significantly up-regulated in the high-risk group than in the low-risk group  therapy, is currently at the forefront of anticancer drugs. The immunoreaction of HCC has emerged as a promising topic worthy of further investigation. Therefore, we analyzed six key immune checkpoint inhibitor genes  in an HCC cohort . The corsk group . There wsk group . These rsk group . In a nuDue to its high morbidity and mortality rate, HCCHCC continues to pose a serious threat to human health. Currently, the prognosis and treatment of HCC patients largely rely on the pathological examination, AJCC TNM, and BCLC stage . The exi+ T cells, activated dendritic cells, central memory CD4+ T cells, effector memory CD4+ T cells, immature dendritic cells, regulatory T cells, type 2 T helper cells Furthermore, the high-risk group also had higher transcriptional levels of HAVCR2 and CTLA‐4 than the low-risk group, indicating that they may have a distinct immune pattern. We also found that the high-risk group had a lower ICI score and a decreased sensitivity to immunotherapy than the low-risk group.This study revealed extensive alterations in pyroptosis genes at the transcriptional, mutation, and CNV levels in HCC. The correlation between pyroptosis genes and HCC prognosis was investigated, and 614 candidate genes were found to be significantly associated with pyroptosis genes at mutation, amplification, and expression levels. Using pathway enrichment analysis, we identified 108 PRGs that were significantly enriched in Hallmark, KEGG, and Reactome pathways. Fifty-seven out of the 108 PRGs exhibited a significant relationship with the prognosis of HCC. Using the LASSO Cox analysis, we constructed a risk score model based on pyroptosis-regulated signaling pathways using seven PRGs, namely UBE2S, KPTN, RNF2, GSR, FTSJ3, DCAF13, and EIF4E. This risk model was evaluated in an external validation cohort LIRI-JP, where it demonstrated a high prediction accuracy. We constructed a nomogram to estimate the survival of patients who were diagnosed with HCC using the risk score model. According to the findings of the ssGSEA analysis, the high-risk group possessed higher levels of activated CD4In 2001, D'Souza et al. proposed the term “pyroptosis”. Since then, many researchers have investigated this novel pro-inflammatory programmed cell death . Due to In the present study, seven genes regulated by pyroptosis, namely UBE2S, KPTN, RNF2, GSR, FTSJ3, DCAF13, and EIF4E were used to establish an independent risk score model. UBE2S or ubiquitin-conjugating enzyme E2 S was found to be up-regulated in HCC . In a to+ T cells and Natural Killer cells (NK)  . Our stuIn summary, our risk score model based on pyroptosis-regulated genes can be used to predict the prognosis of patients, immune cell infiltration characteristics, and response to immunotherapy in patients with HCC.


Legumes, including lentil, are a valuable source of carbohydrates, fiber, protein and vitamins and minerals. Their nutritional characteristics have been associated with a reduction in the incidence of various cancers, HDL cholesterol, type 2 diabetes and heart disease. Among these quality parameters, lectins have been associated with reducing certain forms of cancer, activating innate defense mechanisms and managing obesity. Protease inhibitors such as trypsin and chymotrypsin inhibitors have been demonstrated to reduce the incidence of certain cancers and demonstrate potent anti-inflammatory properties. Angiotensin I-converting enzyme (ACE) inhibitor has been associated with a reduction in hypertension. Therefore, legumes, including lentils, should be part of our daily food intake. However, high temperatures at the terminal stage is a major abiotic constraint leading to a reduction in lentil yield and seed quality. Thus, the selection of heat-tolerant genotypes is essential to identifying the potential for high yields with stable performance. To select lentil genotypes, an experiment was conducted with 60 genotypes including local landraces, advanced breeding lines, commercial varieties and exotic germplasm under stress and non-stress conditions from 2019 to 2020. This study was followed by a subset study involving screening based on a few physicochemical parameters and reproductive traits along with field performances. Different tolerance indices , relative heat index (RHI), tolerance (TOL), mean productivity (MP), stress tolerance index (STI), geometric mean productivity (GMP), yield index (YI), yield stability index (YSI), heat-resistance index (HRI), modified stress-tolerance index (MSTI), abiotic tolerance index (ATI) and stress susceptibility percentage (SSPI)) were used for the selection of the genotypes along with field performance. Biplot analysis was further performed for choosing the most suitable indices. Based on principal components analysis, the GMP, MP, RRI, STI, YI, YSI, ATI and MSTI indices were identified as the most reliable stress indicators, and these indicators might be used for distinguishing heat-tolerant genotypes. Based on the stress indices, the genotypes BLX 05002-3, BLX 10002-20, LRIL-21-1-1-1-1, LRIL-21-1-1-1-1-6 and BLX 09015 were selected as the most stable and heat-tolerant genotypes. In contrast, the genotypes LG 198, Bagura Local, BLX 0200-08-4, RL-12-178, Maitree, 91517 and BLX 11014-8 were selected as the most heat sensitive. Data also exhibited an average yield reduction of 59% due to heat stress on the lentils. Moreover, eight heat-tolerant (HT) genotypes , and two heat-susceptible (HS) genotypes  were selected from the screened genotypes and subjected to further analysis by growing them in the following year under similar conditions to investigate the mechanisms associated with heat tolerance. Comparative studies on reproductive function and physiochemical traits revealed significantly higher pollen viability, proline accumulation, relative water content, chlorophyll concentration and a lower membrane stability index in HT genotypes under heat stress. Therefore, these heat-tolerant genotypes could be used as the parents in the hybridization program for achieving heat-tolerant transgressive segregation. Worldwi−1 . The cen−1 . It is a−1 . Low tem−1 ,5.In Bangladesh, the lentil-sowing date is delayed due to late harvesting of the preceding crop, such as late sown Aman rice in the north-western part of Bangladesh, and early vegetable cultivation is increasing in the western part of Bangladesh. In most cultivated areas, the lentil reproductive stage, especially the grain-filling stage, suffers adversely from elevated temperatures . GeneralA combined package of genetic improvement and cultural practices can help minimize the detrimental effects of various abiotic stresses on agricultural productivity . GeneticA total of 60 lentil genotypes consisting of commercial varieties, local landraces, advanced breeding materials and foreign germplasms were collected from the world’s largest collection center, the International Centre for Agricultural Research in Dry Areas (ICARDA), to be used in this screening program . FollowiThis particular study was carried out at the research farm of PRC, BARI, Ishurdi, Pabna, Bangladesh. The daily maximum and minimum air temperature were recorded on the Zeal Maximum Minimum temperature scale under polythene shades and outside of the experiment to compare the raising temperature under the polythene shades and in control conditions during the crop season 2019–20 and 2020–21. The risIn the 1st year, all collected genotypes were sown under two conditions: (1) one set was sown at the optimum sowing time on 7 November (OS) when day/night temperatures were within 32/8 °C  at theirIn the present study, consisting of sixty genotypes under OS and LS conditions, observations of several parameters were recorded. Days to flowering were counted from the sowing to the point at which 50% of flowers were visible in the plants on a plot basis. The canopy width of randomly selected plants was measured in cm in three positions of the planted rows. The height of five selected plants was measured in cm, representing the average height at the maturity stage. A hundred randomly selected seeds were weighed in grams on a plot basis. For the estimation of grain yield/plant, the whole plot yield divided by the total number of final plant stands was used, before storing the seeds at 8–10% moisture content. The straw yield was the average biomass yield of the five randomly selected plants after oven drying at 72 °C for three days.The harvest index was counted using the following equation:Stress-susceptibility index − Y¯sY¯p The relative heat index YpY¯sY¯p Tolerance (TOL) = Yp − Ys Mean productivity P=Ys+Yp2 Stress-tolerance index + YpY¯p2 Geometric mean productivity GMP=YpYs Yield index YI=YsY¯s Yield-stability index YSI=YsYp Heat-resistance index ×YsYpY¯s 1STI, Modified stress-tolerance index (MSTI) = KAbiotic tolerance index ¯s×Yp×Ys Stress-susceptibility percentage (SSPI) = Y¯p}×100 where Yp, Ys, p and s indicate the yield under normal sowing, yield under late sowing for each genotype, and mean yield in normal and late sowing conditions for all genotypes, respectively. For screening heat-tolerant genotypes, a rank-sum (RS) was calculated using the following relationship [Heat tolerance indices (stress susceptible index (SSI), relative heat index (RHI), tolerance (TOL), mean productivity (MP), stress tolerance index (STI), geometric mean productivity (GMP), yield index (YI), yield stability index (YSI), heat-resistance index (HRI), modified stress-tolerance index (MSTI), abiotic tolerance index (ATI) and stress susceptibility percentage (SSPI)) were estimated for the selection of the heat-tolerant genotypes and these heat-tolerant indices were calculated using the MS Excel program following these formulas:tionship :Rank sumDifferent thermal unit indices, such as growing degree days (GDDs), helio-thermal units (HTU), pheno-thermal index (PTI) and heat-use efficiency (HUE), were calculated at the maturity stage according to Singh et al. . GDDs weHTUs (degree-days hours) of successive growth phases were calculated based on GDD and sunshine hours using the following formula:The duration of sunshine hours per day of successive growth phases was calculated by the following equation:−1) was calculated using the following equation:PTI (degree-days dayHeat-use efficiency (HUE) (kg ha degrees-day) was calculated with the help of the following equation:Some physiological traits, such as proline content, chlorophyll content, relative water content (%), membrane thermal stability index, and one reproductive trait, pollen viability (%), were studied in the following year for further analysis to assess the tolerance mechanism with the association of rising temperature, and methodology of the assessment of these physiochemical traits is described in brief as follows:A = absorbance at a specific wavelength of the spectrophotometer; V = final volume of chlorophyll extract in 80% acetone; W = fresh weight of tissue extractedBefore flowering, 0.1 g of fresh leaves from the control and stress treatment of each genotype were taken in an amber-colored bottle. Following this, 10 mL 80% acetone was mixed with the leaf sample and kept in a dark place for more than 24 h to dissolve the chlorophyll concentration following the procedure of Awasthi et al. . After 2Proline content was detected following the ninhydrin method according to the procedure described by Bates et al. , which fRelative leaf water content (RLWC) was determined following the methods of Barrs and Weatherley . Before 1) of this boiling sample was measured by an electrical conductivity meter. Consequently, another sample of leaves of the same genotype was placed in a water bath for boiling at 100 °C for 10 min and electrical conductivity was recorded in the same way (C2). Finally, MSI was estimated with the following equation:The leaf membrane thermostability index (MSI) was measured following the protocol of Premachandra et al.  as modifDuring the days to flowering of each control and stress genotype, pollen grains were collected from opened flowers on the same days. The collected pollen grains were polled, and the section was prepared based on the size and shape of the pollen and stained with 0.5% acetocarmine/Alexander stain at 10 min . The polAn analysis of variance of different yield and yield-contributing traits, principal component analysis (PCA), and a correlation study were analyzed by a statistical software program . AdditioSixty lentil genotypes were screened based on the field performance of different phenological and yield-contributing traits, such as days to flowering, plant height, canopy width, 100-seed weight, straw yield per plant, grain yield per plant and harvest index. Observations showed that in late-sown environments, all genotypes flowered earlier, ranging from 46 to 67, except genotype 7 (check variety BARI Masur-9) compared with optimum-sown conditions (range 36–73 days for flowering) . Among a−1. In contrast, LS plants ranged from 0.22–1.37 g plant−1 (−1) while the genotypes 44, 57, 24, 47, 46 and 13 produced the minimum GYP (0.222–0.362 g plant−1)  while genotypes 52, 8, 3, 43 and 7 produced the lowest SYP (0.828–1.031 g plant−1). The average harvest index (HI) ranged from 18.86–53.27% estimated in OS plants to 10.25–52.26% in LS plants. Among the genotypes 8 (52.26%), 54 (48.77%), 52 (48.28%), 29 (46.84%), 53 (45.48%) and 43 (41.92) gave the maximum HI, while the genotypes 57, 44, 46, 24, 25 and 40 showed the lowest, with 10.25%, 15%, 15.96%, 16.65%, 18.07% and 18.75% HI, respectively, in a heat-stress environment (The hundred-seed weight (HSW) in OS plants ranged from 1.69–3.34 g, while LS plants ranged from 1.47–3.01 g . In a st plant−1 . In LS cplant−1) . Straw yironment .A wide range of variations in grain size and grain yield was revealed among the studied genotypes in both stress and non-stress environments. In LS plants, gain-size reductions of 1–45% were recorded. The maximum grain-size reduction of 45% was observed in genotype 43; the second-highest, 43%, in genotype 49; and the lowest reduction of 1% was observed in genotypes 12, 27 and 38 . Grain-ySignificant yield variations were observed among all genotypes due to the terminal heat stress .1STI, genotypes 59, 16, 58, 9 and 15—and for K2STI, genotypes 59, 58, 54, 29 and 15—were the most tolerant genotypes relative to the other studied genotypes.The minimum yield reduction was recorded in genotypes 54, 59, 58, 9, 8, 16 and 29 under stress and non-stress environments compared to the other genotypes in the same condition, which means these genotypes were treated as terminal heat-stress tolerant. However, the maximum yield reduction was observed in genotypes 44, 57, 13, 24, 47 and 46. The calculated stress-tolerance attributes  indicateThe above-estimated indices of heat tolerance indicated that, based on a single index, the selection of heat-tolerant genotypes was more contradictory. Different indices indicated different genotypes as heat tolerant. For the determination of the most desirable heat-tolerant genotypes according to all studied indices’ mean rank (MR), the standard deviation of ranks (SDR) and rank-sum (RS) were estimated . With co2STI, TOL, MP, STI, GMP, ATI, SSPI and K1STI . The PC axes separated Yp, TOL, SSPI, ATI, K1STI, MP and GMP in group 2 (G2), and Ys, YSI, RHI, HRI, YI and K2STI in group 3 (G3). “High heat tolerance” should be considered in terms of yield stability in a heat-stress environment. The genotypes that perform their yield with low fluctuations under different stress environments can be considered “highly heat tolerant”.The first two PCAs explained more than 95% of the total variations. The PCA1 and PCA2 categorized the indices into different groups. From these studies, SSI was categorized as group 1 . In contrast, “heat-tolerance” should be considered based on acceptable yield performance under stress conditions and high-yield performance under normal environments and it not refer to the yield stability in both normal and stress environments. Thus, TOL, SSPI, ATI, K1STI, MP, STI and GMP might be considered tools for screening “heat-tolerant” genotypes as they have no relationship with YSI or their negative correlation. The path view of the biplot  association with that of the normal environment  and significant (ironment .p ˂ 0.001), TOL  and SSPI   . These rThe whole life cycle of a lentil is divided into different growth stages, such as seeding, vegetative, reproductive, and mature. The different thermal unit indices were measured at the maturity stage under optimum-sowing (OS), and late-sowing (LS) conditions . The day−1 in OS, whereas in the LS condition this range was recorded from 5.90 to 6.15 h day−1. The helio-thermal units (HTU) of each genotype were recorded at a maximum in the LS condition compared to the OS except for some genotypes, and ranged was 480.43 to 660.51 in OS. These were recorded from 452.53 to 593.11 degree-day hours in LS condition. The pheno-thermal index (PTI) was recorded from 0.68 to 0.95 degree-day day−1 in OS and ranged from 0.82 to 0.97 degree-day day−1 in LS condition. The heat-use efficiency (HUE) of all genotypes under the OS condition was more or less similar (ranging from 41.42 to 42.82) but significant variance was recorded in LS condition and ranged from 5.72 to 30.26. The minimum HUE was recorded in the case of genotype 57, followed by genotypes 47, 46, 45 and 44. The maximum HUE (30.26) was recorded in genotype 33, followed by genotypes 29 (28.32), 59 (26.80), 58 (26.17), 9 (25.59) and 8 (25.44) with higher yield performance in OS and LS conditions.The GDD value is the maximum of each genotype in the LS condition compared to the OS genotypes, except the genotypes 2 and 14. Genotypes 15, 16, 18, 19, 37, 40, 45, 46, 51, 57 and 60 showed higher GDD in OS conditions. The received sunshine hours range was recorded at 6.21 to 6.48 h day−1) was observed in the genotypes BARI Masur-8 followed by the genotypes LRL-21-112-1-1-1-1-6 and BLX 12009-6 and the minimum chlorophyll content 1.61 was observed in the genotype BLX 09015 under the control condition. In heat-stress conditions, the minimum total chlorophyll content of 0.94 mmol g−1 was observed in the genotype PRECOZ and the maximum total chlorophyll content of 1.56 was recorded in the genotype BARI Masur-8.There was no significant variation among the studied genotypes in Chl a, Chl b and total Chl content. However, a reduction in chlorophyll was shown among all genotypes due to heat-stress conditions . NumericAmong the different environmental stress conditions, solutes, such as proline, are normally accumulated in large quantities in higher plants for mitigating stress conditions, such as heat and drought. In this present study, proline content increased significantly under the late-sowing condition compared to the control condition due to the terminal heat-stress environment among all genotypes except the susceptible genotype LG 198. The maximum proline accumulation was found in the heat-tolerant genotype LR-9-25 followed by BLX-05002-6 and BLX-05002-3 and the minimum proline content was recorded in the susceptible genotype BLX 12009-6 .Under the control environment, RWC ranged from 76.67% to 83.67%, which was statistically non-significant, but in the stress condition, significant variations of RWC were recorded among all genotypes. The maximum RWC was recorded in the case of the tolerant genotype BLX 09015, with the minimum reduction percentage (7.17%), followed by the tolerant genotype BLX 05002-6 (7.50% reduction) and LR-9-25 (7.53% reduction) . The higThe membrane thermostability index ranged from 12.00 to 15.53% among the genotypes in the OS condition, which increased from 41.89 to 95.70% due to heat stress in LS condition . The maximum membrane thermostability index (27.73%) was recorded in the susceptible genotype LG-198 with the highest increasing rate (95.70%) followed by another susceptible genotype, BLX 12009-6 (25.67% MSI), with the second-highest increasing rate (84.28%) under the LS condition. The minimum MSI (17.77%) was recorded in the heat-tolerant genotype LRL-21-112-1-1-1-1-6 and followed by the tolerant genotypes BLX 05002-6 (18.10% MSI) and BLX 09015 (18.3%).In the OS condition, the individual genotypes’ pollen viability ranged from 72.04 to 86.62%, which decreased by 3.34–21.60% in the terminal heat-stressed samples among all genotypes . The maxIn this present investigation, we imposed elevated temperatures on lentil genotypes by growing them one month later than the recommended date of sowing as well as covering late-sown experimental field with polythene shades, while a controlled plot was sown at the recommended sowing time for lentil crops. Creating high-temperature stress, delaying sowing, and covering the late-sown crops with polyethene shades is a worldwide recognized technique where the plants are grown in cooler weather to avoid high temperatures and drought stress during their reproductive development phase. However, twelve worldwide stress indices were used for the evaluation of these 60 lentil genotypes against high-temperature stress. Exploiting these indices indicated that the identification of heat-tolerant genotypes was more complex when based on a single index. Different indices indicated different genotypes as heat-tolerant; for instance, RHI and MP were found as suitable indices for identifying relatively more heat-tolerant genotypes, such as the genotypes 54, 48, 31, 58, and 29, and 59, 16, 9, and 15, respectively. However, the stress indices TOL and SSI were used for the selection of the most desirable heat-tolerant genotypes in this study, genotypes 54 and 48. On the other hand, the stress indices STI and GMP were used for screening the most heat tolerant and the least tolerant genotypes relatively, and selected the genotypes 59, 58, 16, 9, 29, 54, 53 and 15 as the most heat tolerant and 44, 13, 24, 47, 57 and 12 as the least relative tolerant genotypes. The index YI also indicated that the genotypes 58, 54, 59 and 29 were the most and 44, 57, 13 and 24 were the least tolerant genotypes relatively. On the other hand, TOl, ATI, SSPI and YI indices can be used for the selection of genotypes based on yield stability in the target environment . For ins0 C higher in LS experimental plots than in OS experimental plots. A very limited set of experiments using the late sowing of lentils covered by polyethene shades has been conducted under field conditions globally. To date, one experiment has been performed by Delahunty et al. [Under field conditions, 60 lentil genotypes were sown one month later than the recommended sowing date and this late-sown experimental plot was covered by polythene shades to artificially create a high temperature. Another control experimental plot was sown at the recommended sowing date with the same genotypes, experimental design and management practices without polyethene shades. The temperature was always 3–4y et al.  in Austry et al.  and chicy et al. ,56,57 byy et al. . −1 was comparative low in the genotypes 58, 10, 35, 2, 4 and 18. Unfilled pods per plant were significantly higher in the OS condition compared to the LS condition in all studied genotypes; this occurred due to maximum pod abortion in the LS environmental condition as a result of the detrimental effects of high temperature in the lentil’s reproductive stage. Early studies showed that reproductive growth is more penetrating and causes various effects, such as the depletion of buds, flowers, fruits, pods and seeds, resulting in marked reductions in yield potential [In this present study, a significant detrimental effect of high temperature was revealed in the case of yield and all yield-contributing traits except on branches per plant in the LS environment. High temperature has a detrimental effect on growth and reproductive physiology due to lentil’s narrow gene pool . Days tootential ,64. A siotential  in lentiotential  also recotential ; whereasotential  or direcotential . The seeotential . Due to otential ,70,71,72otential . Late sootential . In sorgotential ; in mustotential . In commotential .1), Yp, TOL, SSPI, ATI, K1STI, MP and GMP in group 2 (G2), and Ys, YSI, RHI, HRI, YI and K2STI in group 3 (G3). “High heat-tolerance” should be considered in yield stability with low fluctuations under different stress environments. The indices HRI, K2STI, YI and RHI could be used for screening “highly heat-tolerant” genotypes as they have a strong association with YSI (yield-stability index). In contrast, “heat-tolerance” should be considered based on acceptable yield performance under stress conditions and high yield performance under normal environments and it should not refer to the yield stability in both normal and stress environments. Thus, TOL, SSPI, ATI, K1STI, MP, STI and GMP could be considered tools for screening “heat-tolerant” genotypes as they have no relationship with YSI or their negative correlation. The path view of the biplot represents a summary of the interrelationships among the heat indicators and also provides the importance of the indices, which is more or less emphasized for the identification of the individual index of the specific stress. Principal component analysis (PCA) showed that a significant positive correlation was observed between grain yield in stress conditions with the criteria TOL, SSPI, ATI, YP, K1STI, MP and GMP; consequently, they can distinguish heat-tolerant genotypes with the same approach. Exploring these indices for the selection of heat-tolerant genotypes in lentils is very rare globally but very common in durum and bread wheat [PCA is exploited by plant breeders as a “pattern finding method” for completing cluster analysis . The PCAad wheat ,46 for tad wheat . Mohammaad wheat  and Saread wheat  also repad wheat  for the 1STI and K2STI. Thus, these indices could be used for the identification of high-heat-tolerance genotypes based on the stress-tolerance yield performance of the 60 studied lentil genotypes. Many researchers have used multicollinearity better ascertain relationships between interpretive traits and yield performance [Multicollinearity tests are used as a powerful tool by plant breeders to find whether multicollinearity exists between measured traits or indices ,82. The formance ,84.1STI and K2STI were the most significant and reliable selection indicators for the screening of stress-tolerant genotypes among the 60 studied genotypes of lentil. A similar finding was recorded by Puri et al. [A correlation study among the studied indices revealed that GMP, MP, RDI, STI, YI, YSI, DI, ATI, Ki et al.  in sprinTemperature, as well as sunshine hours, are significant and basic weather elements for any crop’s growth and development. For development from one growth stage to another, the crop needs a specific time and amount of heat . In the present study, the 60 lentil genotypes were evaluated based on the different thermal unit indices with their days to maturity and yield performance in OS and LS conditions. In the case of yield performance, each genotype produced a higher yield in the OS condition compared to the LS condition. All studied genotypes showed a higher GDD in LS condition except genotypes 2, 14, 15, 16, 18, 19, 37, 40, 45, 46, 51, 57 and 60. The received sunshine duration of each genotype in hours was maximal in OS compared to LS conditions. The helio-thermal units (HTU), and the pheno-thermal index (PTI) were recorded at their maximum in OS compared to LS conditions for each genotype. The heat-use efficiency (HUE) was more or less stable in each genotype under the OS condition but in LS conditions the HUE declined in the case of all genotypes except genotypes 29, 33, 58, 59, 9 and 8 due to their higher yield performance. Genotypes 29, 33, 58, 59, 9 and 8 were identified as heat-tolerant genotypes based on the thermal unit indices, whereas genotypes 57, 47, 46, 45 and 44 were selected as heat-sensitive genotypes due to their lower yield performance. Similar research was conducted by Islam et al.  in the cThe stability of the chlorophyll concentration in plant cells during stress conditions is reported as a potential tool for selection against stressful environments in peanuts  and pigeThe genotype LR-9-25 accumulated a higher proline content followed by the genotypes BLX-05002-6 and BLX-05002-3, and the minimum concentration was recorded in the case of the susceptible genotype BLX-12009-6. However, proline accumulations of the studied genotypes were found to be at their maximum in the stress environment compared to those sown at the optimum time. These results clearly indicate that the genotypes LR-9-25, BLX 05002-6 and BLX 05002-3 are more tolerant than the genotypes BLX 12009-6 and LG-198 owing the higher proline concentration under stress conditions. A similar finding was observed by Stoyanov  in beansThe relative water content (RWC) is the percentage of water held by plant tissues. Generally, RWC is at a maximum in plant tissue in control environments compared to stress conditions. From this study, no significant variations were observed in any lentil genotypes during the control condition, but all genotypes exhibited a significant reduction in relative water content during the terminal heat-stress condition, and this reduction was significantly greater in susceptible genotypes than in the tolerant ones. A similar result was observed by Dash et al. . The redElevated temperature stress affects a plant’s normal physiological parameters adversely, such as membrane stability, photosynthesis, respiration and protein metabolism, which exacerbates low productivity in growth and yield . SeveralThe reproductive stages, especially the flowering and grain-filling stages, are more susceptible to elevated temperature stress; a few degrees of rise in temperature may lead to complete crop loss during the pollen-development stage ,106,107.1STI and K2STI have been identified as the most significant and reliable selection indicators for the evaluation of heat-tolerant genotypes based on comparing the performance of the 60 lentil genotypes under stress and non-stress environments. With consideration of all the indices, genotypes 54(BLX 10002-20), 29(BLX 05002-6), 58(BLX 05002-3) and 8(LRIL-21-67-1-1-1-1) were selected as the most heat-tolerant genotypes. In contrast, genotypes 44 , 24 (BLX 0200-8-4), 47 (Maitree), 13 (91517), 57 (LG 198) and 45 (RL-12-178) were identified as the most sensitive to heat-stress environments among all the studied genotypes. Simultaneously, considering the field performance under stress and non-stress environments indicated that the genotypes 54 (BLX 10002-20), 58 (BLX 05002-3) and 29 (BLX 05002-6) showed the maximum harvest index, with a minimum yield reduction of 22, 38 and 39%, respectively, in the heat-stress environment. In contrast, genotypes 57(LG 198), 40 (BLX 12004-5) and 44  showed the minimum harvest index with the maximum yield reductions of 82, 79, and 72%, respectively. The average yield loss due to heat stress in lentils was recorded at 59% in this present investigation.Stress indices are successfully used for the identification of superior genotypes with tolerant genetic resources and better agronomical performance in a stressful environment. However, twelve stress indices were explored in this present investigation and among these indices, GMP, MP, RDI, STI, YI, YSI, DI, ATI, K


Uterine leiomyomas usually arise from the uterine body (95%), and rarely from the cervix (0.6%) or other urogenital sites. Lipoleiomyomas are benign, uncommon variants of leiomyomas (0.03–0.2%), histologically composed of smooth muscle cells and mature adipocytes; they usually occur in the uterine body and exceptionally in the cervix. We performed the first systematic literature review of cervical lipoleiomyomas (PRISMA guidelines), presenting five new cases. Including our series, thirty-one detailed cases were reported in the literature (mainly in Asia). The age range was 35–74 years, revealing a higher mean age than conventional cervical leiomyomas (46.5 vs. 39.4 years). Patients were usually multiparous (94%), typically complaining of vaginal bleeding , pelvic/abdominal pain , and/or urinary disturbances  1 week to 10 months before presentation. Clinical examination revealed a pedunculated tumor (48%), or prolapse of ≥1 pelvic organs (16%). Twenty-four (77%) patients underwent total hysterectomy ± additional surgery; simple myomectomy/excision was performed in five (16%) cases. Only one (3%) of our cases recurred 2 years after partial excision; no evidence of disease was found 13 years after recurrence excision. Adipocytes occupied ≤50% of the tumor volume. Hyaline or myxoid changes and cartilaginous metaplasia were uncommon histological findings. Surgically challenging cases or pregnant patients may require expert gynecologists. Interventional radiology or conservative treatments were rarely proposed. Uterine leiomyomas are benign smooth muscle tumors, typically found in the uterine body (95% of cases), with a prevalence of 20–40% after 35 years of age ,7,8. PedLipoleiomyomas (LLMs) are rare histological variants of leiomyomas, being composed of smooth muscle cells mixed with variable extension of mature adipocytes; they account for about 0.03–0.2% of all uterine leiomyomas ,12,13,14In the present study, we report a new series of five cervical LLMs; in addition, we have performed the first SLR on these tumors to better delineate their clinicopathologic features, imaging results, treatment, and follow-up.Five cases of cervical LLMs were retrieved from our files. The specimens had been fixed in 10% buffered formalin and routinely processed. Formalin-fixed, paraffin-embedded blocks were sectioned, and the resulting slides were stained with hematoxylin and eosin. The following immunohistochemical markers were tested in case 2: smooth muscle actin  and CD31 .http://www.prisma-statement.org/; accessed on 15 October 2022)  guidelines (er 2022)  to identPopulation: human patients with a diagnosis of LLM of the cervix;Intervention: any;Comparison: none;Outcomes: patients’ clinical outcomes .Our retrospective observational study was conducted through the PICO process: https://pubmed.ncbi.nlm.nih.gov, accessed on 15 October 2022), Scopus  and Web of Science  databases. We also searched for “lipoleiomyoma” plus “cervix” or “cervical” in the Google Scholar database . No limitations or additional filters were set. The bibliographic research ended on 15 October 2022. We applied the following:Eligibility/inclusion criteria: studies reporting LLMs of the cervix in human patients.Exclusion criteria: unclear diagnosis; LLMs of other sites; non-analyzable results (aggregated data).We searched for ((lipoleiomyoma OR lipomatous OR lipoleiomyomatous OR (leiomyoma AND (fat OR adipose OR fatty))) AND cervix) in the Pubmed . After applying the eligibility, inclusion, and exclusion criteria, they selected 28 relevant eligible papers, which were all obtained in full-text format and screened for additional references. After reading the full-texts, three articles were excluded as they were unfit according to the inclusion/exclusion criteria (too aggregated data) [Two independent authors removed the duplicates and checked the titles and abstracts of all the retrieved results (ed data) ,16,17. Ted data) ,40,41,42A 43-year-old woman (P6402) presented with vaginal spotting. The speculum exam revealed a regular cervix with an evident “cystic” lesion on the anterior cervical lip. On transvaginal ultrasounds, the lesion was supposed to be a 2.3-cm cervical leiomyoma; a posterior subserosal leiomyoma of the uterine corpus was also identified. No other relevant findings were reported. The patient was regularly followed up but developed progressive uterine prolapse. Three years later, the patient’s condition worsened, revealing a 3rd-degree uterine prolapse and moderate cysto-rectocele; the woman complained of stress incontinence. A Pap smear resulted in a negative. Total vaginal hysterectomy by morcellement, bilateral salpingectomy, and pelvic floor repair were performed. The uterus was split into three fragments, two of the uterine corpus  and one of the elongated, hypertrophic cervix (length: 6 cm). On gross examination, a 2.1 cm grayish-yellow intramural nodule with mainly regular margins was identified in the wall of the anterior cervix .A few other grayish nodules were found in the myometrium of the uterine corpus: the bigger nodule measured 1.6 cm in maximum size. On histological examination , the cerNecrotic areas, nuclear atypia, mitoses, hemorrhage, regressive changes, vascular proliferation, heterologous components, and vascular invasion were not identified. A diagnosis of cervical LLM was made. The other nodules in the uterine corpus were conventional leiomyomas. A proliferative endometrium and superficial adenomyosis were also reported. The Fallopian tubes were regular, showing a right paratubal serous cystadenoma (diameter: 2.5 cm). No evidence of recurrence was found after 1 year.A 43-year-old woman (P3003) underwent total abdominal hysterectomy for multiple uterine leiomyomas and adhesiolysis due to three previous caesarean sections and bilateral tubal sterilization (5 years before). The uterus weighed 225 gr and measured 12 × 9 × 8 cm. Two grayish fascicular intramural leiomyomas were found in the uterine corpus, the bigger of which measured 4.2 cm in diameter. A small LLM (cm 0.8) was incidentally found in the uterine cervix; adipocytes accounted for about 45% of the tumor volume . No necrotic areas, mitoses, or nuclear atypia were present. On immunohistochemical analysis , smooth A 42-year-old woman with a 2nd–3rd degree cystocele showed an endometrial polyp and a nodular lesion protruding from the cervix. The nodule was excised, and an endometrial polypectomy was performed. The nodule was well-delimited, grayish, elastic, and fascicular, measuring 3.8 cm in diameter. On histological examination, it revealed a submucosal LLM of the uterine cervix; the adipocytes accounted for about 25% of the tumor volume. No necrosis, nuclear atypia, or mitoses were found. An endometrial polyp was also diagnosed. No evidence of recurrence was found after 17 years.In 2007, a 40-year-old woman without previous significant history underwent hysteroscopy for currently unavailable symptoms; the uterine cavity was regular without mucosal lesions in the endometrium or endocervix, while a hard, sessile, but partially twisted exocervical polypoid lesion of 2 cm in maximum size was identified and partially excised. On histological examination, the polyp was revealed to be a cervical LLM. After 2 years, a pedunculated polypoid gray lesion was found to have grown and protruded from the cervix (maximum size: 5 cm), probably representing a “recurrence” of the previously partially removed LLM. After excision, histological examination confirmed a cervical submucosal LLM. In both specimens, nuclear atypia, necrosis, mitoses, and other worrisome features were absent, and the adipocytic component covered less than 10% of the tumor volume. Thirteen years later, no evidence of disease was found on a gynecological examination. In 2010, a 44-year-old woman underwent total hysterectomy and bilateral salpingo-oophorectomy for a right ovarian cystic multilocular Sertoli cell tumor of 8 cm in maximum size . A 1-cm polypoid endocervical nodule was also identified; on histological examination, it was revealed to be a cervical LLM without necrosis, nuclear atypia, or mitoses. The adipocytes accounted for about 5% of the tumor volume ; after sGlobally, including our series, only 44 cases of cervical LLMs were found in the literature ,40,41,42Akbulut et al. described 76 LLMs (2.9% of the uterine LLMs in their files) arising in the uterine corpus , cervix , retroperitoneum (one case), and broad ligament (one case) .Bolat et al. found 10 (1.4%) LLMs among 707 uterine leiomyomas; only 1/10 (10%) of these cases arose in the cervix .Wang et al. reported a series of 50 LLMs occurring in the cervix , uterine corpus , retroperitoneum (one case), and broad ligament (one case) .As reported in the Materials and Methods, we had to exclude the following three series from further analysis because the clinicopathologic features were not reported in detail for each case:So, including our cases, only 31 detailed cases of cervical LLMs were reported in the literature, according to our systematic literature review  18,19,2,40,41,42The age range of the reported 31 patients was 35–74 years . Ten women 32%) were clearly or probably postmenopausal ,40,41,42% were clThe most frequent symptoms were vaginal bleeding 11/31, 36%) ,36,39,41% ,36,39,The time interval between the onset of symptoms and the clinical exam ranged from 1 week to 10 months. Thirteen patients 42%) complained of one symptom ,39,40,41% complaiFifteen patients (48%) showed a mass/polyp that was described as protruding through the vagina, pedunculated, and/or increased in size ,36,40,42Globally, 5/31 (16%) patients experienced a prolapse of pelvic organs (PPO) ,28,40. In = 10, 32%) [n = 1, 3%) [n = 1, 3%), 2.1-cm adult granulosa cell tumor of the right ovary  [n = 1, 3%) [Other findings included synchronous leiomyomas of the uterine body  ,30,34,40= 1, 3%) , 8-cm Se= 1, 3%) , and cer= 1, 3%) ,42.On ultrasound examination, the tumors were described as heterogeneous/complex echoic  ,33,38, hComputed tomography scans revealed intralesional fat in two cases (6%) ,42. The Magnetic resonance imaging information was available for three patients (10%): in two cases (6%), these LLMs were hyperintense on T1/T2 scans and heterogeneously hypointense on fat-suppressed T2 scans ,23, whilTwenty-four (77%) patients underwent total hysterectomy  ,39,40,41Simple myomectomy or excision of the nodule protruding through the vagina were performed in five (16%) cases ,29,42; 1Follow-up information was reported in 16/31 cases (52%), despite frequently being incomplete: post-operative complications were not reported in any case ,40,41,42When data were available, the LLMs were located in the anterior  ,20,28,29Four LLMs (13%) were located near to the isthmus, including a tumor also extending to the parametrial tissues ,26,35,38On gross examination, the nodules were solid, usually well-circumscribed ,40,41,42n = 50; including seven cervical cases), the distribution of adipocytes widely varied, showing a mean value of 36% [n = 10; including one cervical case), adipocytes occupied 5% to 95% of the tumors [n = 76; including five cervical cases) classified the LLMs according to the extension of intratumor adipocytes as grade 1  , grade 2 (moderate) , or grade 3 tumors   [When data were available, adipocytes occupied ≤50% of the tumor volume in the cases analyzed in our review. Three studies were excluded from our analysis as they reported data on cervical LLMs aggregated with those of LLMs arising in the uterine body or extrauterine sites ,16,17. I38, 50%) . One cer38, 50%) . Another38, 50%) .Nuclear atypia, mitotic figures, and necrotic areas were absent in all the cases, including our recurrent LLM. Thick-walled blood vessels were occasionally described, without vascular proliferation.Limited information is available about the immunohistochemical profile of cervical LLMs, as immunohistochemistry was performed in only 11/31 (35%) cases ,37,40,41The proliferation (Ki-67/Mib-1) index was ≤1% in both the smooth muscle and adipocytic components of the three tested cases ,26,37. Molecular analysis was not performed on cervical LLMs. To the best of our knowledge, we are here presenting the largest Italian and European series of cervical LLMs and the first SLR on this topic.The histogenesis of LLMs is still unknown, including the following hypotheses: lipoblastic differentiation of misplaced embryonic progenitor cells; metaplastic changes of connective or smooth muscle tissue into adipocytes; perivascular adipocytes; traumatic displacement of fat tissue; pluripotent cell migration along the uterine nerves and vessels and fatty infiltration ,15. In the normal cervical stroma, smooth muscle fibers—from which leiomyomas or LLMs can arise—are more typical in the endocervix than in the exocervix. It is also controversial if mature adipocytes are normal or heterotopic constituents of the cervical stroma in women of various ages  ,44. BeinPure malignant or benign lipomatous tumors (liposarcomas or lipomas) or other rare mesenchymal tumors with occasional foci of adipocytic differentiation (such as solitary fibrous tumors) also enter the differential diagnosis with LLMs; they are usually reported in the uterine body, exceptionally occurring in the cervix ,50,51,52Clinical guidelines are increasingly recommending that gynecological cancers should be treated in specialized centers by expert gynecologic surgeons and oncologists; however, the surgical treatment of benign tumors can also be challenging ,54,55,56Some authors suggested that preoperative gonadotropin-releasing hormone (GnRH) agonists (23% of CLs), the tourniquet method, intraoperative injection of vasopressin-epinephrine into the myometrium 72% of CLs), and permanent (POUA) or temporary (TOUA) occlusion of the uterine artery  could reduce the risk of bleeding during CL-myomectomy 2% of CLs,61,62,63n = 214), excluding the LLMs analyzed by our study due to a different research method; treatment approaches included surgery , interventional radiology treatment (IRT) , surgery + IRT , and exclusive conservative management . Surgical procedures comprised myomectomy , hysterectomy , and trachelectomy . Only 7/124 (5.6%) patients with available data experienced post-operative complications after laparoscopic myomectomy  [A recent systematic literature review  focused e fever) ,73,74, le fever) , laparose fever) , or vagie fever) . In our nt (IRT) /214, 9%,omy n = 1, 67.5%,  n = 54, .7%, and e fever) ,40,41,42The mean age of our patients with cervical LLMs was slightly higher, compared to the CLs of Ferrari et al. (46.5 vs. 39.4 years); we probably included more postmenopausal women , but the fertility status was available for only 57/214 (27%) patients in the review of Ferrari et al. ,40,41.The prevalence of uterine leiomyomas in pregnant women ranges approximately from 3% to 10%, but CLs are even rarer . CLs canFerrari et al. identified only 23 (40%) pregnant patients and one (2%) puerperal patient with CL; 20/23 87%) women were treated conservatively during their pregnancy, including patients who subsequently underwent surgery  or were still conservatively treated  after delivery % women w. MoreoveOnly three (13%) patients with CL were treated with surgery  or IRT (1 super-selective uterine fibroid embolization) during pregnancy ,64,80,81Despite the still limited results, IRT can be promising for the treatment of CLs, especially in cases of surgical contraindications or the patient’s desire for uterine preservation. These women may benefit from super-selective embolization of the leiomyoma or cervicovaginal artery; however, subsequent pregnancies after uterine embolization have a statistically significantly higher rate of spontaneous abortion (56% vs. 10.5%), malpresentation (20%), and cesarean section 80%) compared to those following surgical uterine artery occlusion . In the % compareLong-term follow-up was rarely available for CLs or LLMs ,40,41,42Morcellement may favor tumor recurrence; iatrogenic parasitic leiomyomas (IPLs) may result from morcellated leiomyomas that have implanted far from the uterus, developing an independent blood supply from adjacent structures. The overall incidence of IPLs after laparoscopic surgery using a morcellation approach accounts for 0.12–0.95% (0.20–1.25% after laparoscopic myomectomy). The time interval between the initial surgery and diagnosis ranges from 3 to 8 years ,87. OnlyMalignant transformation of leiomyomas/lipoleiomyomas is exceptional; morcellation of an unsuspected leiomyosarcoma might worsen the prognosis ,89,90. Tn = 2; 6%), vaginal prolapse , cystocele , and/or rectocele  [In our series, 5/31 (16%) patients experienced a PPO, showing a variable association of uterine prolapse (UP)  ,28,40. I= 2; 6%) ,94,95,96= 2; 6%) ,98. Glob= 2; 6%) ,98. Well= 2; 6%) ,100. Pro= 2; 6%) . Pedunculated submucosal uterine leiomyomas prolapsing through the cervical canal can mimic a UP, although some studies suggest their potential role in causing UP/PPO ,102,103.In younger women of childbearing age, hypertrophic cervical elongation can mimic UP because it has a similar presentation, but it lacks other anatomical defects (such as descent of the uterus and level 1 ligament impairment) . AlthougCompared to leiomyomas of the uterine body, CLs were rarely described in literature; LLMs represent a rare variant of uterine leiomyomas, and only 31 cases were found to arise from the cervix. Vaginal bleeding, pelvic pain, urinary disturbances, and a tumor protruding through the cervical os are the main complaints; 5/31 (16%) patients were associated with a PPO, while 3/31 (10%) cases showed an elongated/distorted cervix, potentially playing a role in their pathogenesis by dragging down the pelvic organs (as to the tumor size and weight). Myomectomy or hysterectomy is the therapy of choice based on the desire for pregnancy and anatomic features; however, surgery may be challenging, requiring expert gynecologists. Interventional radiology or conservative treatments have rarely been proposed to manage CLs; further studies are required.


To explore the carrying status and homology of carbapenem resistant Acinetobacter baumannii (CRAB) in our hospital.From January 2015 to December 2017, 52 strains of acinetobacter baumannii isolated from the bacteria room of the clinical laboratory of Baogang hospital in Inner Mongolia were selected as the research object. K-B disk diffusion method and Vitek-2 were used to determine the drug sensitivity of Acinetobacter baumannii. The drug resistance gene was detected by polymerase chain reaction (PCR) and its homology was analyzed by pulsed field gel electrophoresis (PFGE).Except for Cefoperazone/sulbactam, other antibiotics were resistant to ab. The detection rate of drug resistance gene class C β-lactamases (ADC) was 100%, and the higher detection rates of other drug resistance genes were class D β-lactamases (OXA)-51 ,disinfectant gene qacE△1-sull , and klebsiella pneumoniae carbapenemase (KPC) gene was not detected. 2-8 drug resistance genes were detected in each CRAB strain, and the strains with 6 drug resistance genes were the most ; Among the detected drug-resistant gene combinations, ADC+OXA-23 + OXA-51 gene was detected at the same time , followed by ADC+ intl1 + qacE △ 1-sull gene , ADC + qacE △ 1-sull + ant (3 ‘‘) -i gene , and 11 strains (27.5%). There were 19 different types in PFGE homology test, each type was 1-9 strains, including 9 strains of A5 type and 8 strains of A18 type, mainly from intensive care unit.CRAB in the hospital is highly resistant to common clinical antibiotics. OXA-23 and OXA-51 genes are most likely to be the main factors causing drug resistance of Acinetobacter baumannii in the hospital. Homology analysis showed that there was CRAB nosocomial infection transmission in different wards of the hospital. Acinetobacter baumannii (AB) is a conditional pathogen , accountThe drug resistance mechanism of AB is very complex and can be mediated by a variety of drug resistance mechanisms. If the expression of outer membrane protein changes, it can cause the barrier of antibiotic permeability; Changes of topoisomerase and DNA gyrase; Overexpression of efflux pump of various drugs . This pa40 CRAB strains isolated from the bacteria room of the clinical laboratory of Inner Mongolia Baogang hospital from January 2015 to December 2017 were selected as the research objects. Remove duplicate strains isolated from the same patient. The isolated strains were identified and tested by automatic microbial identification and drug sensitivity instrument. Some drug sensitivity tests and reviews were conducted by disk diffusion method (K-B method). The drug sensitivity results were judged according to the American Society for clinical laboratory standardization (CLSI) version 2021. The quality control strains were Escherichia coli ATCC25922, Pseudomonas aeruginosa atcc27853, enzyme producing Escherichia coli atcc35218 and Klebsiella pneumoniae atcc70060.PCR FQZD (BIOER), nucleic acid electrophoresis apparatus , automatic gel imaging analysis system , fluorescence quantitative polymerase chain reaction (PCR) instrument , electronic constant temperature stainless steel water bath pot .RNA extraction reagent and DNA loading buffer were purchased from cwbio company. Dnamarker DL2000, reverse transcription PCR kit and Supermix were purchased from tran company. Agarose and dyes were purchased from xhly company.The susceptibility of the strains to 15 kinds of antibiotics was detected by disk diffusion method (K-B method). Antibacterial drugs include: Cefoperazone/sulbactam, cefotaxime, ceftriaxone, levoflOXAcin, tobramycin, ceftazidime, imipenem, meropenem, piperacillin, piperacillin/tazobactam, gentamicin, amikacin, cefepime, minocycline, compound minocycline, etc. The pieces of paper were purchased from oxid. Columbia blood tablet was purchased from French merier company, and MH tablet was purchased from Zhengzhou Beiruite company. The judgment of drug sensitivity results shall be carried out according to the 2021 standard of American Society for clinical laboratory standardization (CLSI) .PFGE homology detection: after CRAB was isolated and cultured, the bacteria suspension with OD value of 3.6 ~ 4.0 was adjusted by turbidimeter. Add 1%seakem gold SDS to prepare gel. Digest with protease K, wash with pure water for 2 times, and then wash with TE for 4 times, about 15min each time. ApaI endonuclease was used for enzyme digestion and incubated in 37°C water bath for 4H. PFGE was performed in a pulsed field gel electrophoresis apparatus. The electrophoretic parameters were 5 ~ 20s, 14°C, 120° pulse angle electrophoresis for 19h. After electrophoresis, nucleic acid staining was performed, which was put into a gel apparatus for observation and photo preservation.Eight strains of multidrug-resistant bacteria were selected as the drug-resistant group, and the gene expression levels of the strains in the drug-resistant group and the sensitive group were detected.Extract the strain RNA according to the operation steps of the bacterial RNA extraction kit of Omega company, use the nucleic acid protein analyzer to detect the concentration of RNA and carry out electrophoresis. The genes were amplified by realtime PCR. The reaction system is 20ul, including 0.4ul of upstream and downstream primers and 5 UL of cDNA template, 2 × PerfectStartTM Green qPCR Super Mix10uL. Make up the volume to 20ul with DD H2O. The amplification conditions were three-step, the first step was 95 °C, 30s; 1 cycle; The second step is 95 °C, 5S; 60 °C, 30s, 40 cycles. See Import the images saved by the gel imager into the bionumerily software for processing and analysis, and calculate the similarity coefficient between the strains with the Dice coefficient . SD=2nxyA total of 40 CRAB strains were collected from 40 patients. The sources of specimens were vascular catheter tip (9 strains), drainage fluid (5 strains), blood (5 strains), bronchial lavage fluid (5 strains), urine (4 strains), secretion (4 strains), sputum (4 strains), cerebrospinal fluid (2 strains), ascites (1 strain), bile (1 strain), and 28 strains of CRAB were from intensive care unit (ICU). See Among them, 40 AB strains were resistant to cefotaxime, ceftriaxone, levoflOXAcin, tobramycin, ceftazidime, imipenem, meropenem, piperacillin, piperacillin/tazobactam, gentamicin, amikacin, cefepime and minocycline, and the resistance rate was 100%. 10 AB strains were sensitive to Cefoperazone/sulbactam, and the resistance rate was 25.0%. 36 AB strains were sensitive to cotrimOXAzole, and the resistance rate was 90.4%. See ADC gene was detected in all 40 CRABs. The detection rates of other drug resistance genes were OXA-51 (90.0%), qacE△1-sull (80.0%), OXA-23 (77.5%), intl1 (72.5%), and KPC gene was not detected. 2-8 drug resistance genes were detected in each CRAB strain, and the strains with 6 drug resistance genes were the most (37.5%); Among the drug-resistant gene combinations detected, ADC + OXA-23 + OXA-51 gene was the most detected at the same time , followed by ADC + intl1 + qacE△1-sul1 gene , ADC + qacE△1-sull + ant (3″) -1 gene , ADC + OXA-23 + OXA-51 + intl1 + qacE△1-sull gene , and 11 strains (27.5%). ADC+ant (3″) -i + ACC(3) -i gene was detected. See 40 CRAB PFGE clustering map showed that the number of electrophoretic bands ranged from 22 to 29, as shown in Acinetobacter baumannii is an aerobic gram-negative conditional pathogen, which widely exists in the hospital environment. Due to its strong viability and resistance, the infection of Acinetobacter baumannii has become a difficult problem to be solved in hospitals all over the world. Due to the irrational use of antibiotics and the lack or inadequate implementation of nosocomial infection management and control measures, the drug resistance of bacteria is on the rise. Multidrug resistant AB has become the main pathogen of nosocomial infection  and spreProduced by bacteria β- Lactamases are mainly divided into four categories, and category a mainly refers to broad spectrum β- Lactamases, including TEM, SHV, KPC, etc; Class B metal β- Lactamases, including imp, vim and sim; Class C is amp C enzyme; Class D is OXAcillinase, also known as OXAcarbapenemase, of which OXA-23 is the most common OXAcillinase in China. Various β-The combined action of lactamases can mediate the resistance of Acinetobacter baumannii to penicillin, carbapenems, monocyclic amides and cephalosporins . In thisThe detection rates of OXA-51 and OXA-23 were 90.0% and 77.5% respectively. The detection rate of OXA-23 gene was basically similar to that reported in the domestic literature , and no The detection rate of disinfectant resistance gene qacE△1-sull was 80.0%. At present, the most studied disinfectant resistance gene is quaternary ammonium disinfectant resistance gene, namely qac gene. The qac gene family includes qac A, qac B, qac C, qac d, qacE, qac F, etc. . It is aIn this study, the number of CRAB strains is relatively small, and CRAB resistant to imipenem and meropenem has not been studied. Some cases of increased sensitivity of AB to imipenem may be omitted due to the deletion of OXA-23 gene . More saThis study showed that the main drug resistance genes of CRAB were ADC, OXA-51 and OXA-23. TEM, ADC, OXA-51 and OXA-23 genotypes are higher in CRAB, which is the main cause of β-lactam resistance. Homology study showed that there was a small range of drug resistance gene clone transmission in Acinetobacter baumannii in our hospital. Hospital infection control monitoring should be actively carried out to prevent CRAB clone transmission. We should reasonably use antibacterial drugs, strengthen the improvement of existing antibacterial drugs and the research and development of new antibacterial drugs.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Inner Mongolia Baogang hospital.H-BG conceived of the study, and H-LH participated in its design and coordination, and Y-YL helped to draft the manuscript. All authors read and approved the final manuscript.


Dictyostelium cells. Actin assembled de novo at the wound site, independent of cortical flow. Next, we searched for actin- and signal-related proteins targeting the wound site. Fourteen of the examined proteins transiently accumulated at different times. Thirdly, we performed functional analyses using gene knockout mutants or specific inhibitors. Rac, WASP, formin, the Arp2/3 complex, profilin, and coronin contribute to the actin dynamics. Finally, we found that multiple signaling pathways related to TORC2, the Elmo/Doc complex, PIP2-derived products, PLA2, and calmodulin are involved in the actin dynamics for wound repair.The repair of wounded cell membranes is essential for cell survival. Upon wounding, actin transiently accumulates at the wound site. The loss of actin accumulation leads to cell death. The mechanism by which actin accumulates at the wound site, the types of actin-related proteins participating in the actin remodeling, and their signaling pathways are unclear. We firstly examined how actin accumulates at a wound site in  Dictyostelium cells [2+ influx from an external medium is essential for wound repair. The “membrane patch hypothesis” has been proposed to explain wound pore plugging, wherein cytosolic membrane vesicles accumulate at the wound site and fuse to form an impermanent “patch” to plug the wound pore as an emergency reaction [The cell membrane is a barrier between the extracellular and intracellular spaces, but it is consistently subjected to wounding by physical or chemical damage. However, wounded cell membranes can be repaired. Loss of wound repair is seen in various diseases such as muscular dystrophy ,2. The mum cells . A commoreaction ,14,15. Treaction . Variousreaction ,17,18. HXenopus eggs and Drosophila embryos, an actomyosin ring, similar to the contractile ring in dividing cells, surrounds the wound site in the cell membrane, and its constriction facilitates the closure of the wound pore [Dictyostelium cells, only actin transiently accumulates at the wound site [Dictyostelium cells, mammalian culture cells, and muscle cells [Dictyostelium cells, and the deletion of myosin II does not affect wound repair [In large cells, such as und pore ,19,20. Hund site ,21,22,23le cells ,24,25. Ile cells ,22,23,26d repair . Similard repair ,29.The mechanism by which actin accumulates to form specific actin structures in specific cortical regions, such as pseudopodia and filopodia, has been intensively investigated. Active Rac and Cdc42, small Rho-family GTPases, activate Wiskott–Aldrich Syndrome protein (WASP) family proteins, such as WASP, SCAR, and WASH. In turn, these WASP family proteins activate the Arp2/3 complex, which generates a meshwork of branched F-actin . Cdc42 aIn addition to Arp2/3 and formin, many species of actin-related proteins (ARPs) regulate actin organization and its dynamics, and are categorized as crosslinking, monomer-binding, bundling, stabilizing, branch-forming, and membrane-binding . To form2+ and calmodulin regulate actin assembly in Dictyostelium cells [Xenopus oocytes and yeast during wound repair [Drosophila embryos and Xenopus oocytes [As mediators of signaling pathways for wound-induced actin assembly, WASP, Arp2/3, and formin have been implicated in regulating actin assembly ,34,35,36um cells . Proteind repair ,37. Rho  oocytes ,38. In a oocytes ,39.Dictyostelium cells as well as neutrophils have been intensively investigated [Dictyostelium, to its membrane receptors, which are trimeric G protein-coupled receptors (GPCRs), causes actin assembly at the leading edge to extend pseudopods. Several pathways are involved in actin assembly and chemotaxis, including phosphatidylinositol 3,4,5-trisphosphate kinase (PI3K)-, phospholipase A2 (PLA2)-, cGMP-, TORC2-, and Elmo/Doc complex-mediated pathways, which seem to form a cross-talk network [The signaling pathways of actin assembly in the chemotaxis of stigated ,41. The  network ,43,44. AIn this study, we examined how actin accumulates at a wound site and the types of ARPs that participate in actin remodeling. With reference to the signal cascades for chemotaxis, we also examined the types of signals that regulate the dynamics of these proteins and found several candidates for the signaling pathways. This information should also provide insights into the mechanism not only for actin organization during wound repair but also for those during many actin-dependent cellular events such as chemotaxis, phagocytosis, macropinocytosis, and cell adhesions.Dictyostelium discoideum wild-type (AX2) and mutant cells were cultured at 22 °C in a plastic dish containing HL5 medium , as previously described [2, and 3 mM MES, pH 6.3), and the cells were incubated in the same solution for 5–6 h.escribed . For wouDescriptions of the plasmid sources and null mutant cells used in the present study are listed in We constructed expression vectors for some GFP-tagged proteins, including RacA, severin, filamin, ABD34, CAP32, fimbrin, α-actinin, and CARMIL. The individual genes were amplified from a cDNA library by PCR and subcloned between the BamH1 and Sac1 sites downstream of the C-terminal GFP site in the pA15GFP expression vector, as described previously .The surface of the coverslip of a glass-bottom chamber was coated with gold using a sputtering device , in place of carbon vapor deposition, as previously described ,48. The http://rsbweb.nih.gov/ij, accessed on 1 September 2022). The fluorescence intensities were normalized by setting the value before wounding to 1 after subtracting the background.Cells expressing GFP-tagged proteins were observed under a total internal reflection fluorescence (TIRF) microscope , as previously described . The celTo observe the influx of PI, a 0.15 mg/mL propidium iodide (PI) solution  was added to the external medium before wound experiments. PI fluorescence was monitored by illumination with an argon laser (488 nm) under TIRF microscopy, as described before .v/v), which did not affect wound repair, as described before [Latrunculin A, jasplakinolide, W7 hydrochloride, CK666, CK869, SMIFH2, EHT1864, wiscostatin, Torin 1, U73122, and LY294002 were purchased from Funakoshi Co., Ltd. . BPB was purchased from the FUJIFILM Wako Pure Chemical Corporation . Thiabendazole was purchased from Tokyo Chemical Industry Co., Ltd. . All inhibitors were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions and kept at −30 °C until use. Cells were incubated with the following concentrations of inhibitors: 1 µM latrunculin A, 8 µM jasplakinolide, 20 µM W7 hydrochloride, 60 µM CK666, 50 µM CK869, 10 µM SMIFH2, 50 µM EHT1864, 10 µM wiscostatin, 50 µM Torin 1, 4 µM U7312220, 20 µM LY294002, 2 µM BPB, and 100 µM thiabendazole. Inhibitors were used at a higher than IC50 concentration. The IC50 concentrations of CK666, U731220, BPB, wiscostatin, and torin1 were based on macropinocytosis inhibition in previous report . The cond before . Wound et-test for comparison between two groups, or one-way ANOVA with Tukey’s multiple comparisons test.Statistical analyses were performed using GraphPad Prism 7 . Data were expressed as means ± SD and analyzed using a two-tailed Student’s We previously reported a novel method for creating wounds in cell membranes using laserporation ,46. In tIt was easy to focus the laser spot on the surface of the coverslip. When the laser beam was thus focused, the gold coating peeled off and appeared as a small white spot B. BecausDictyostelium, a model organism for studying cell migration, chemotaxis, and cell division. To examine the opening of a pore in the cell membrane by laserporation, PI, which emits fluorescence upon binding to RNA or DNA, was added to the external medium (BSS). We used the improved laserporation method to examine the wound repair mechanism in Dictyostelium cells [We have shown that actin transiently accumulates at the wound site in um cells ,23,47. Fum cells . Previouum cells ,53,54,55um cells ,56. FiguTherefore, actin accumulation at the wound site is essential for wound repair.Xenopus oocytes, the actomyosin ring is generated by the flow of cortical actin filaments toward the wound site, accompanied by dynamic actin polymerization [There are two possible mechanisms for actin accumulation at a wound site: (1) preexisting cortical actin filaments flow  to accumulate toward the wound site (flow model), and (2) actin polymerizes at the wound site (de novo synthesis model). In rization ,57. HoweTo verify the de novo synthesis model, cells expressing GFP-lifeact were wounded in the presence of latrunculin A and/or jasplakinolide, a stabilizer of actin filaments A. As desNext, when cells were incubated with jasplakinolide for 30 min and then with latrunculin A, large and small actin aggregates were observed. Under these conditions, the number of polymerizable actin monomers should be significantly reduced, although the pre-built actin structures such as the aggregates are stabilized by jasplakinolide. Upon wounding, actin did not accumulate at the wound site  was monitored using cells expressing GCaMP6s, a Ca2+ sensor. Neither showed any positive response, although Ca2+ levels transiently increased in the cytosol  participate in proper temporal and spatial regulation. To clarify the types of ARPs that contribute to actin remodeling, cells expressing various GFP-tagged ARPs were wounded. Many types of ARPs transiently accumulated at the wound site at different times A. SuppleRemarkably, severin (an F-actin-severing protein similar to gelsolin) accumulated at the wound site almost immediately. WASP (a protein that initiates actin polymerization), Arp3 (a component of the Arp2/3 complex that nucleates actin filaments activated with WASP), ABP34 (an actin-bundling protein), and MyoB (a type I myosin) accumulated significantly earlier than actin. Alpha-actinin (an actin-crosslinking protein), filamin (an actin-crosslinking protein), and CAP32 (an actin capping protein) began to accumulate simultaneously with actin. MyoC (a type I myosin), CARMIL (a multidomain scaffold protein), and fimbrin (an actin-bundling protein) began to accumulate after actin. Coronin and cofilin accumulated in the actin disassembly stage, consistent with the previous consensus that coronin inactivates the Arp2/3 complex  and thatInterestingly, EfaA1  transiently disappeared from the wound site , SCAR (a member of the WASP family), vinculin (involved in cell–substrate adhesion by interacting with actin), GmfA (an inactivator of Arp2/3), paxillin (involved in cell–substrate adhesion by interacting with actin), formin A (an actin-nucleation protein), and DwwA (a ww-domain-containing protein) did not accumulate at the wound site .In summary, specific types of ARPs may accumulate with their unique temporal and spatial regulations to remodel actin structures at wound sites.To examine the ARPs required for wound-induced actin dynamics, knockout mutants of these proteins were transformed with GFP-lifeact, and the response of actin accumulation upon wounding was examined. The mutants available in DictyBase and NBRP Nenkin stock centers were mainly used. Several inhibitors were also used. Although severin immediately accumulated at the wound site as described above, actin normally accumulated at the wound site in severin-null cells . WASP-nuWe have shown that myosin II transiently disappears from the wound site . NeverthProfilin A/B double-null cells significantly reduced the amplitude of actin accumulation and retarded termination . These mOther knockout mutants, including filamin-null, α-actinin/filamin double-null, WASH-null, and cortexillin A/B double-null cells, showed responses similar to those of wild-type cells .Dictyostelium cells.Overall, these results indicate that WASP, Arp2/3, formin, profilin, and coronin are required for proper actin dynamics during wound repair in Dictyostelium as well as neutrophils have been intensively investigated [Supplementary Figure S4.Signal cascades for chemotaxis in stigated ,66,67,682+ induces Ca2+ release from the endoplasmic reticulum, mediated by inositol 1,4,5-triphosphate (IP3) receptor-like protein A (IplA), which is homologous to IP3 receptors [i2+ responses than wild-type cells [The influx of Caeceptors . Woundedpe cells . As expeDouble-knockout mutants of GcA/SgcA (membrane-bound and soluble-guanylyl cyclases) and GbpC/GbpD (cGMP-binding proteins) showed an actin accumulation curve similar to that of wild-type cells , indicatPI3K is a key regulator of actin polymerization in chemotactic responses ,71. The In contrast, GFP-PTEN (phosphatase and tensin homolog deleted from chromosome 10), which converts PIP3 to phosphatidylinositol 3,4-bisphosphate (PIP2), transiently disappeared from the wound site , and thep-bromophenacyl bromide, BPB) significantly retarded the peak and termination times of actin dynamics (PLA2 (phospholipase A2) is one of the signaling proteins involved in chemotaxis, and acts parallel to the PI3K pathway ,73. A PLdynamics . The Elmdynamics , suggestDictyostelium, is involved in chemotaxis [The TORC2 also plays a critical role in chemotaxis . Activatemotaxis ,81. An iOverall, the pathways involving PIP2-derived products, PLA2, the Elmo/Dock complex, and TORC2 mediate wound-induced actin dynamics.Dictyostelium cells have 20 Racs but no Rho and Cdc42 [The Rho family of small G proteins is an upstream signal for regulating actin dynamics during chemotaxis and may be shared with wound-induced actin dynamics. nd Cdc42 . A Rac iRasG and RasC, members of the Ras family of small G proteins, are critical mediators of chemotactic responses . RasG acDictyostelium, did not affect actin accumulation (P21-activated protein kinases (Paks) act on a large number of regulators of the actin cytoskeleton in higher eukaryotes, including RhoGDI, LIM kinase, filamin, diverse myosin kinases, the Arp2/3 complex, and paxillin . Howevermulation . The ortmulation .The serine/threonine kinase, Phg2, binds Ras GTPases and PIP2 to regulate phagocytosis, macropinocytosis, cell–substrate adhesion, cytokinesis, and actin filament organization ,86. The Cyclase-associated protein (CAP) is an important regulator of cell polarity, chemotaxis, and cell adhesion, and is linked to the actin cytoskeleton . GFP-CAPDictyostelium, did not affect actin accumulation , followed by the second peak (25–40 s) [When 25–40 s) ,98,99. T2+-dependent actin-severing protein, we first hypothesized that it may cut and remove actin filaments that are damaged or disordered by wounding. However, we did not observe any removal of cortical actin filaments in wild-type cells, and the actin dynamics in severin-null cells were similar to those in wild-type cells.Our experiments using GFP-tagged proteins showed that specific types of ARPs accumulate at wound sites with different times to remodel actin structures. Surprisingly, severin immediately accumulates at the wound site. Since severin is a CaDictyostelium [WASP, Arp2/3, formin, and ABP34 accumulated earlier at the wound site than actin. Although we did not examine the effects of the inhibition of ABP34, the inhibition of WASP, Arp2/3, or formin retarded both the initiation and termination times of actin accumulation and reduced the amplitude, suggesting that these proteins contribute to actin assembly at the wound site. Although the Arp2/3 complex and formins have been considered to play different roles in actin regulation, their regulation may be partially synergistic . The forostelium ,101,102.2+ in vitro, accumulate at the wound site. However, knockout mutants (ABP34-null was not determined) did not affect actin accumulation. Incidentally, single-knockout mutants of these ABPs show no distinct phenotypes of cell migration, chemotaxis, or cell division in Dictyostelium [Several ABPs, such as α-actinin, fimbrin, severin, and ABP34, which are regulated by Caostelium .Coronin and cofilin accumulate at the wound site in the stage of actin disassembly. Although we could not inhibit cofilin because it is essential and null mutants are not available , coroninChemotaxis signaling pathways were used as a reference. Among these, the cAMP and cGMP pathways did not mediate wound-induced actin accumulation. PI3K pathways did not mediate actin accumulation either, although PI3K signaling via RasG/C and PKB is one of the key regulators of actin dynamics for chemotaxis . Indepen2+-dependent manner. The PLA2 pathway is involved in chemotaxis in parallel to the PI3K pathway [i2+ [The PLA2 pathway is also a candidate, because this specific inhibitor retarded the termination of actin accumulation. PLA2 generates arachidonic acid-related lipids in an intracellular Ca pathway . In addiway [i2+ . The ElmDictyostelium cells have 20 Racs, the exact Rac responsible for wound-induced actin accumulation needs to be identified in the future.The Rac inhibitor retarded both the initiation and termination times of actin dynamics and reduced the amplitude, suggesting that a member of the Rac family contributes to actin accumulation. Presumably, such a Rac may be activated by RacGEF and then activates WASP, Arp2/3, and formin to induce actin assembly. Since In the present study, we found that calmodulin is located upstream of annexin C1 and actin. The counterpart of calmodulin is yet to be identified. Incidentally, there are many calmodulin-binding proteins, including those related to actin regulation, such as DwwA, Htt, RgaA, MHCKs A and B, Paks A and B, GapA, RacGEF, CaM-BP46, and MyoG . The con2+ enters the cytosol upon wounding, calmodulin and annexin C1 accumulate immediately at the wound site, which finally induces actin assembly. The TORC2, Dock/Elmo, PIP2-derived product, and PLA2 pathways are common in chemotaxis and wound repair. Racs, WASPs, and then formins and Arp2/3 are involved in these pathways, and further downstream, many ARPs regulate the actin dynamics at the wound site. We believe that these pathways can be completed by combining multiple gene-knockout mutants and inhibitors in the future. Since the induction of local actin assembly by wounding is highly reproducible, the laserporation method is a powerful tool to obtain general and useful information on actin dynamics during other actin-related phenomena such as chemotaxis, phagocytosis, and cell–substrate adhesion.We have summarized the signaling pathways for actin dynamics in wound repair in