Correction: Magnaporthe oryzae fimbrin organizes actin networks in the hyphal tip during polar growth and pathogenesis.

[This corrects the article DOI: 10.1371/journal.ppat.1008437.].

Maintaining calcium homeostasis as a strategy to alleviate nephrotoxicity caused by evodiamine.

Evodiamine (EVO), the main active alkaloid in Evodia rutaecarpa, was shown to exert various pharmacological activities, especially anti-tumor. Currently, it is considered a potential anti-cancer drug due to its excellent anti-tumor activity, which unfortunately has adverse reactions, such as the risk of liver and kidney injury, when Evodia rutaecarpa containing EVO is used clinically. In the present study, we aim to clarify the potential toxic target organs and toxicity mechanism of EVO, an active monomer in Evodia rutaecarpa, and to develop mitigation strategies for its toxicity mechanism. Transcriptome analysis and related experiments showed that the PI3K/Akt pathway induced by calcium overload was an important step in EVO-induced apoptosis of renal cells. Specifically, intracellular calcium ions were increased, and mitochondrial calcium ions were decreased. In addition, EVO-induced calcium overload was associated with TRPV1 receptor activation. In vivo TRPV1 antagonist and calcium chelator effects were observed to significantly reduce body weight loss and renal damage in mice due to EVO toxicity. The potential nephrotoxicity of EVO was further confirmed by an in vivo test. In conclusion, TRPV1-mediated calcium overload-induced apoptosis is one of the mechanisms contributing to the nephrotoxicity of EVO due to its toxicity, whereas maintaining body calcium homeostasis is an effective measure to reduce toxicity. These studies suggest that the clinical use of EVO-containing herbal medicines should pay due attention to the changes in renal function of patients as well as the off-target effects of the drugs.

The Microtubule End Binding Protein Mal3 Is Essential for the Dynamic Assembly of Microtubules during Magnaporthe oryzae Growth and Pathogenesis.

Cytoskeletal microtubules (MTs) play crucial roles in many aspects of life processes in eukaryotic organisms. They dynamically assemble physiologically important MT arrays under different cell conditions. Currently, aspects of MT assembly underlying the development and pathogenesis of the model plant pathogenic fungus Magnaporthe oryzae (M. oryzae) are unclear. In this study, we characterized the MT plus end binding protein MoMal3 in M. oryzae. We found that knockout of MoMal3 results in defects in hyphal polar growth, appressorium-mediated host penetration and nucleus division. Using high-resolution live-cell imaging, we further found that the MoMal3 mutant assembled a rigid MT in parallel with the MT during hyphal polar growth, the cage-like network in the appressorium and the stick-like spindle in nuclear division. These aberrant MT organization patterns in the MoMal3 mutant impaired actin-based cell growth and host infection. Taken together, these findings showed that M. oryzae relies on MoMal3 to assemble elaborate MT arrays for growth and infection. The results also revealed the assembly mode of MTs in M. oryzae, indicating that MTs are pivotal for M. oryzae growth and host infection and may be new targets for devastating fungus control.

The actin motor protein OsMYA1 associates with OsExo70H1 and contributes to rice secretory defense by modulating OsSyp121 distribution.

Magnaporthe oryzae (M. oryzae) is a devastating hemibiotrophic pathogen. Its biotrophic invasive hyphae (IH) are enclosed in the extrainvasive hyphal membrane produced by plant cells, thus generating a front line of the battlefield between the pathogen and the host plants. In plants, defense-related complexes such as proteins, callose-rich materials and vesicles, are directionally secreted to this interface to confer defense responses, but the underlying molecular mechanism is poorly understood. In this study, we found that a Myosin gene, Myosin A1 (OsMYA1), contributed to rice defense. The OsMYA1 knockout mutant exhibited decreased resistance to M. oryzae infection. OsMYA1 localizes to the actin cytoskeleton and surrounds the IH of M. oryzae. OsMYA1 interacts with an exocyst subunit, OsExo70H1, and regulates its accumulation at the plasma membrane (PM) and pathogen-plant interface. Furthermore, OsExo70H1 interacted with the rice syntaxin of the plants121 protein (OsSyp121), and the distribution of OsSyp121 to the PM or the pathogen-plant interface was disrupted in both the OsMYA1 and OsExo70H1 mutants. Overall, these results not only reveal a new function of OsMYA1 in rice blast resistance, but also uncover a molecular mechanism by which plants regulate defense against M. oryzae by OsMYA1-initiated vesicle secretory pathway, which originates from the actin cytoskeleton to the PM.

Immune regulation and prognosis indicating ability of a newly constructed multi-genes containing signature in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common renal malignancy, although newly developing targeted therapy and immunotherapy have been showing promising effects in clinical treatment, the effective biomarkers for immune response prediction are still lacking. The study is to construct a gene signature according to ccRCC immune cells infiltration landscape, thus aiding clinical prediction of patients response to immunotherapy. METHODS: Firstly, ccRCC transcriptome expression profiles from Gene Expression Omnibus (GEO) database as well as immune related genes information from IMMPORT database were combine applied to identify the differently expressed meanwhile immune related candidate genes in ccRCC comparing to normal control samples. Then, based on protein-protein interaction network (PPI) and following module analysis of the candidate genes, a hub gene cluster was further identified for survival analysis. Further, LASSO analysis was applied to construct a signature which was in succession assessed with Kaplan-Meier survival, Cox regression and ROC curve analysis. Moreover, ccRCC patients were divided as high and low-risk groups based on the gene signature followed by the difference estimation of immune treatment response and exploration of related immune cells infiltration by TIDE and Cibersort analysis respectively among the two groups of patients. RESULTS: Based on GEO and IMMPORT databases, a total of 269 differently expressed meanwhile immune related genes in ccRCC were identified, further PPI network and module analysis of the 269 genes highlighted a 46 genes cluster. Next step, Kaplan-Meier and Cox regression analysis of the 46 genes identified 4 genes that were supported to be independent prognosis indicators, and a gene signature was constructed based on the 4 genes. Furthermore, after assessing its prognosis indicating ability by both Kaplan-Meier and Cox regression analysis, immune relation of the signature was evaluated including its association with environment immune score, Immune checkpoint inhibitors expression as well as immune cells infiltration. Together, immune predicting ability of the signature was preliminary explored. CONCLUSIONS: Based on ccRCC genes expression profiles and multiple bioinformatic analysis, a 4 genes containing signature was constructed and the immune regulation of the signature was preliminary explored. Although more detailed experiments and clinical trials are needed before potential clinical use of the signature, the results shall provide meaningful insight into further ccRCC immune researches.

PEDF Protects Endothelial Barrier Integrity during Acute Myocardial Infarction via 67LR.

Maintaining the integrity and protecting the stability of tight junctions in endothelial cells is a potential therapeutic strategy against myocardial ischaemia. Laminin receptors (67LR) are highly expressed on endothelial cell membranes and are associated with endothelial barrier function. Herein, we sought to demonstrate the direct effects of pigment epithelial-derived factor (PEDF) on tight junctions between endothelial cells via 67LR during acute myocardial infarction (AMI) and elucidate its underlying mechanisms. We detected that PEDF directly increased the level of the tight junction protein zonula occludens protein 1 (ZO-1) after overexpression in vitro and in vivo using Western blotting. Evans Blue/TTC staining showed that PEDF significantly reduced the size of the infarcted myocardium. Immunofluorescence and the transwell cellular experiments suggested that PEDF significantly upregulated PI3K-AKT permeability and the distribution of ZO-1 between endothelial cells under OGD conditions. Interestingly, PEDF significantly upregulated the phosphorylation levels of PI3K-AKT-mTOR under oxygen and glucose deprivation conditions but had no significant effects on the total protein expression. The protective effect of PEDF on ZO-1 was significantly inhibited following the inhibition of PI3K-AKT-mTOR. The activation of phosphorylation of PI3K-AKT-mTOR by PEDF was blocked after silencing 67LR, as were the protective effects of PEDF on ZO-1. Therefore, we have reason to believe that PEDF increased ZO-1 expression through the 67LR-dependent PI3K-AKT-mTOR signaling pathway, thus maintaining tight junction stability and protecting cardiac function.

Magnaporthe oryzae fimbrin organizes actin networks in the hyphal tip during polar growth and pathogenesis.

Magnaporthe oryzae causes rice blast disease, but little is known about the dynamic restructuring of the actin cytoskeleton during its polarized tip growth and pathogenesis. Here, we used super-resolution live-cell imaging to investigate the dynamic organization of the actin cytoskeleton in M. oryzae during hyphal tip growth and pathogenesis. We observed a dense actin network at the apical region of the hyphae and actin filaments originating from the Spitzenkörper (Spk, the organizing center for hyphal growth and development) that formed branched actin bundles radiating to the cell membrane. The actin cross-linking protein Fimbrin (MoFim1) helps organize this actin distribution. MoFim1 localizes to the actin at the subapical collar, the actin bundles, and actin at the Spk. Knockout of MoFim1 resulted in impaired Spk maintenance and reduced actin bundle formation, preventing polar growth, vesicle transport, and the expansion of hyphae in plant cells. Finally, transgenic rice (Oryza sativa) expressing RNA hairpins targeting MoFim1 exhibited improved resistance to M. oryzae infection, indicating that MoFim1 represents an excellent candidate for M. oryzae control. These results reveal the dynamics of actin assembly in M. oryzae during hyphal tip development and pathogenesis, and they suggest a mechanism in which MoFim1 organizes such actin networks.

Magnaporthe oryzae Transcription Factor MoBZIP3 Regulates Appressorium Turgor Pressure Formation during Pathogenesis.

The devastating fungus Magnaporthe oryzae (M. oryzae) forms a specialized infection structure known as appressorium, which generates enormous turgor, to penetrate the plant cells. However, how M. oryzae regulates the appressorium turgor formation, is not well understood. In this study, we identified MoBZIP3, a bZIP transcription factor that functioned in pathogenesis in M. oryzae. We found that the pathogenicity of the MoBZIP3 knockout strain (Δmobzip3) was significantly reduced, and the defect was restored after re-expression of MoBZIP3, indicating that MoBZIP3 is required for M. oryzae virulence. Further analysis showed that MoBZIP3 functions in utilization of glycogen and lipid droplets for generation of glycerol in appressorium. MoBZIP3 localized in the nucleus and could bind directly to the promoters of the glycerol synthesis-related genes, MoPTH2, MoTGL1 and MoPEX6, and regulate their expression which is critical for glycerol synthesis in the appressorium turgor pressure generation. Furthermore, the critical turgor sensor gene MoSln1 was also down regulated and its subcellular localization was aberrant in Δmobzip3, which leads to a disordered actin assembly in the Δmobzip3 appressorium. Taken together, these results revealed new regulatory functions of the bZIP transcription factor MoBZIP3, in regulating M. oryzae appressorium turgor formation and infection.

[Protective effect of ophiopogonin D against isoproterenol-induced cardiomyocyte injury and targets].

This study aims to unveil the effect of ophiopogonin D(OPD) on isoproterenol(ISO)-induced apoptosis of rat cardiomyocytes and the possible targets, which is expected to provide clues for further research on the myocardial protection of ophiopogonins. Cell count kit-8(CCK-8) assay was used to detect viability of cells treated with OPD and ISO, Western blot to examine the effect of OPD and ISO on the expression of endoplasmic reticulum stress-related Bip, Bax, Perk, ATF4, caspase-12, and CHOP, flow cytometry to determine cell apoptosis rate, and Hoechst 33258 and Tunel staining to observe cell apoptosis and morphological changes. In addition, the probe for calcium ion-specific detection was employed to investigate calcium ion release from the endoplasmic reticulum, and OPD-bond epoxy-activated agarose solid-phase microspheres were prepared and used as affinity matrix to capture OPD-binding target proteins in H9 c2 cell lysate. For the target proteins of OPD identified by high-resolution mass spectrometry, the related signal pathways were enriched and the potential targets of OPD against cardiomyocyte injury were discussed. The experimental result showed that 10 µmol·L~(-1) ISO can significantly induce the expression of endoplasmic reticulum stress-related proteins and promote cell apoptosis. Different concentration of OPD can prevent the damage of myocardial cells caused by ISO. According to mass spectrometry results, 19 proteins, including Fam129 a and Pdia6, were involved in multiple signaling pathways such as the unfolded protein reaction bound by the ERN1 sensor, tricarboxylic acid cycle, and Nrf2 signal transduction pathway. The above results indicate that OPD protects cardiomyocytes by regulating multiple signaling pathways of target proteins and affecting cell cycle progression.

Bioinformatic analysis identifying FGF1 gene as a new prognostic indicator in clear cell Renal Cell Carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) has been the commonest renal cell carcinoma (RCC). Although the disease classification, diagnosis and targeted therapy of RCC has been increasingly evolving attributing to the rapid development of current molecular pathology, the current clinical treatment situation is still challenging considering the comprehensive and progressively developing nature of malignant cancer. The study is to identify more potential responsible genes during the development of ccRCC using bioinformatic analysis, thus aiding more precise interpretation of the disease METHODS: Firstly, different cDNA expression profiles from Gene Expression Omnibus (GEO) online database were used to screen the abnormal differently expressed genes (DEGs) between ccRCC and normal renal tissues. Then, based on the protein-protein interaction network (PPI) of all DEGs, the module analysis was performed to scale down the potential genes, and further survival analysis assisted our proceeding to the next step for selecting a credible key gene. Thirdly, immunohistochemistry (IHC) and quantitative real-time PCR (QPCR) were conducted to validate the expression change of the key gene in ccRCC comparing to normal tissues, meanwhile the prognostic value was verified using TCGA clinical data. Lastly, the potential biological function of the gene and signaling mechanism of gene regulating ccRCC development was preliminary explored. RESULTS: Four cDNA expression profiles were picked from GEO database based on the number of containing sample cases, and a total of 192 DEGs, including 39 up-regulated and 153 down-regulated genes were shared in four profiles. Based on the DEGs PPI network, four function modules were identified highlighting a FGF1 gene involving PI3K-AKT signaling pathway which was shared in 3/4 modules. Further, both the IHC performed with ccRCC tissue microarray which contained 104 local samples and QPCR conducted using 30 different samples confirmed that FGF1 was aberrant lost in ccRCC. And Kaplan-Meier overall survival analysis revealed that FGF1 gene loss was related to worse ccRCC patients survival. Lastly, the pathological clinical features of FGF1 gene and the probable biological functions and signaling pathways it involved were analyzed using TCGA clinical data. CONCLUSIONS: Using bioinformatic analysis, we revealed that FGF1 expression was aberrant lost in ccRCC which statistical significantly correlated with patients overall survival, and the gene's clinical features and potential biological functions were also explored. However, more detailed experiments and clinical trials are needed to support its potential drug-target role in clinical medical use.

IMUP and GPRC5A: two newly identified risk score indicators in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic cancer has been a threateningly lethal malignant tumor worldwide. Despite the promising survival improvement in other cancer types attributing to the fast development of molecular precise medicine, the current treatment situation of pancreatic cancer is still woefully challenging since its limited response to neither traditional radiotherapy and chemotherapy nor emerging immunotherapy. The study is to explore potential responsible genes during the development of pancreatic cancer, thus identifying promising gene indicators and probable drug targets. METHODS: Different bioinformatic analysis were used to interpret the genetic events in pancreatic cancer development. Firstly, based on multiple cDNA microarray profiles from Gene Expression Omnibus (GEO) database, the genes with differently mRNA expression in cancer comparing to normal pancreatic tissues were identified, followed by being grouped based on the difference level. Then, GO and KEGG were performed to separately interpret the multiple groups of genes, and further Kaplan-Meier survival and Cox Regression analysis assisted us to scale down the candidate genes and select the potential key genes. Further, the basic physicochemical properties, the association with immune cells infiltration, mutation or other types variations besides expression gap in pancreatic cancer comparing to normal tissues of the selected key genes were analyzed. Moreover, the aberrant changed expression of key genes was validated by immunohistochemistry (IHC) experiment using local hospital tissue microarray samples and the clinical significance was explored based on TCGA clinical data. RESULTS: Firstly, a total of 22,491 genes were identified to express differently in cancer comparing to normal pancreatic tissues based on 5 cDNA expression profiles, and the difference of 487/22491 genes was over eightfold, and 55/487 genes were shared in multi profiles. Moreover, after genes interpretation which showed the > eightfold genes were mainly related to extracellular matrix structural constituent regulation, Kaplan-Meier survival and Cox-regression analysis were performed continually, and the result indicated that of the 55 extracellular locating genes, GPRC5A and IMUP were the only two independent prognostic indicators of pancreatic cancer. Further, detailed information of IMUP and GPRC5A were analyzed including their physicochemical properties, their expression and variation ratio and their association with immune cells infiltration in cancer, as well as the probable signaling pathways of genes regulation on pancreatic cancer development. Lastly, local IHC experiment performed on PAAD tissue array which was produced with 62 local hospital patients samples confirmed that GPRC5A and IMUP were abnormally up-regulated in pancreatic cancer, which directly associated with worse patients both overall (OS) and recurrence free survival (RFS). CONCLUSIONS: Using multiple bioinformatic analysis as well as local hospital samples validation, we revealed that GPRC5A and IMUP expression were abnormally up-regulated in pancreatic cancer which associated statistical significantly with patients survival, and the genes' biological features and clinical significance were also explored. However, more detailed experiments and clinical trials are obligatory to support their further potential drug-target role in clinical medical treatment.

Prognostic significance of TOP2A in non-small cell lung cancer revealed by bioinformatic analysis.

BACKGROUND: Lung cancer has been a common malignant tumor with a leading cause of morbidity and mortality, current molecular targets are woefully lacking comparing to the highly progressive cancer. The study is designed to identify new prognostic predictors and potential gene targets based on bioinformatic analysis of Gene Expression Omnibus (GEO) database. METHODS: Four cDNA expression profiles GSE19188, GSE101929, GSE18842 and GSE33532 were chosen from GEO database to analyze the differently expressed genes (DEGs) between non-small cell lung cancer (NSCLC) and normal lung tissues. After the DEGs functions were analyzed, the protein-protein interaction network (PPI) of DEGs were constructed, and the core gene in the network which has high connectivity degree with other genes was identified. We analyzed the association of the gene with the development of NSCLC as well as its prognosis. Lastly we explored the conceivable signaling mechanism of the gene regulation during the development of NSCLC. RESULTS: A total of 92 up regulated and 214 down regulated DEGs were shared in four cDNA expression profiles. Based on their PPI network, TOP2A was connected with most of other genes and was selected for further analysis. Kaplan-Meier overall survival analysis (OS) revealed that TOP2A was associated with worse NSCLC patients survival. And both GEPIA analysis and immunohistochemistry experiment (IHC) confirmed that TOP2A was aberrant gain of expression in cancer comparing to normal tissues. The clinical significance of TOP2A and probable signaling pathways it involved in were further explored, and a positive correlation between TOP2A and TPX2 expression was found in lung cancer tissues. CONCLUSION: Using bioinformatic analysis, we revealed that TOP2A could be adopted as a prognostic indicator of NSCLC and it potentially regulate cancer development through co-work with TPX2. However, more detailed experiments are needed to clarify its drug target role in clinical medical use.

Fimbrin associated with Pmk1 to regulate the actin assembly during Magnaporthe oryzae hyphal growth and infection.

The dynamic assembly of the actin cytoskeleton is vital for Magnaporthe oryzae development and host infection. The actin-related protein MoFim1 is a key factor for organizing the M. oryzae actin cytoskeleton. Currently, how MoFim1 is regulated in M. oryzae to precisely rearrange the actin cytoskeleton is unclear. In this study, we found that MoFim1 associates with the M. oryzae mitogen-activated protein (MAP) kinase Pmk1 to regulate actin assembly. MoFim1 directly interacted with Pmk1, and the phosphorylation level of MoFim1 was decreased in Δpmk1, which led to a change in the subcellular distribution of MoFim1 in the hyphae of Δpmk1. Moreover, the actin cytoskeleton was aberrantly organized at the hyphal tip in the Δpmk1, which was similar to what was observed in the Δmofim1 during hyphal growth. Furthermore, phosphorylation analysis revealed that Pmk1 could phosphorylate MoFim1 at serine 94. Loss of phosphorylation of MoFim1 at serine 94 decreased actin bundling activity. Additionally, the expression of the site mutant of MoFim1 S94D (in which serine 94 was replaced with aspartate to mimic phosphorylation) in Δpmk1 could reverse the defects in actin organization and hyphal growth in Δpmk1. It also partially rescues the formation of appressorium failure in Δpmk1. Taken together, these findings suggest a regulatory mechanism in which Pmk1 phosphorylates MoFim1 to regulate the assembly of the actin cytoskeleton during hyphal development and pathogenesis.

Positive benefit-risk ratio of Psoraleae Fructus: Comprehensive safety assessment and osteogenic effects in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (PF), the dried fruit of Psoralea corylifolia L., is a commonly used traditional medicine that has contributed to the treatment of orthopedic diseases for thousands of years in China. However, recent PF-related liver injury reports have drawn widespread attention regarding its potential hepatotoxicity risks. AIM OF THE STUDY: This study was aimed to evaluate the long-term efficacy and chronic toxicity of PF using a 26-week administration experiment on rats in order to simulate the clinical usage situation. MATERIALS AND METHODS: The PF aqueous extract was consecutively administrated to rats daily at dosages of 0.7, 2.0, and 5.6 g/kg (equivalent to 1-8 times the clinical doses for humans) for as long as 26 weeks. Samples were collected after 13, 26, and 32 weeks (withdrawal for 6 weeks) since the first administration. The chronic toxicity of PF was evaluated by conventional toxicological methods, and the efficacy of PF was evaluated by osteogenic effects in the natural growth process. RESULTS: In our experiments, only the H group (5.6 g/kg) for 26-week PF treatment demonstrated liver or kidney injury, which the injuries were reversible after 6 weeks of withdrawal. Notably, the PF treatment beyond 13 weeks showed significant benefits for bone growth and development in rats, with a higher benefit-risk ratio in female rats. CONCLUSIONS: PF displayed a promising benefit-risk ratio in the treatment and prevention of osteoporosis, a disease that lacks effective medicine so far. This is the first study to elucidate the benefit-risk balance associated with clinical dosage and long-term use of PF, thereby providing valuable insights for rational clinical use and risk control of PF.

Successful Treatment of Multiple Plantar and Periungual Warts by Local Hyperthermia Treatment: A Case Report.

Local hyperthermia treatment, a common immunoadjuvant therapy, has been used in treating various human papillomavirus (HPV) infections. For multiple warts, it can clear off all warts of the body through targeted irradiation on one wart. We reported a case of a 22-year-old patient with multiple plantar warts combined with periungual warts, in which previous treatments were ineffective. Therefore, we utilized local hyperthermia treatment at 44°C using the largest fused-wart plaque on the right plantar as the treatment target for 30-minute irradiation each time. The hyperthermia induction therapy was performed on day 1, 2, 3, 12 and 13, followed by weekly maintenance therapy for 6 times. After 2 months of treatment, both irradiated and non-irradiated lesions disappeared, and no recurrence occurred during the follow-up period. This case indicates that local hyperthermia treatment may be a safe and effective approach for patients with multiple, recalcitrant, and site-specific viral warts due to the ineffectiveness of conventional treatment.

YAP/STAT3 inhibited CD8+ T cells activity in the breast cancer immune microenvironment by inducing M2 polarization of tumor-associated macrophages.

BACKGROUND: Breast cancer (BC) is the leading cause of cancer-related death among women. One of the hallmarks of cancer is sustained angiogenesis. YAP/STAT3 may promote angiogenesis and driving BC progression. This study aimed to investigate how YAP/STAT3 affects the immune microenvironment in BC and understand the underlying mechanism. METHODS: To establish a tumor-associated macrophages (TAMs) model, macrophages were cultured in the 4T1 cell culture medium. A BC mouse model was created by injecting 4T1 cells. The expression of YAP, STAT3, p-STAT3, VEGF, VEGFR-2, and PD-L1 was analyzed using immunofluorescence, western blotting, and quantitative real-time PCR. Flow cytometry was used to identify M1 and M2 macrophages, CD4+ T, CD8+ T, and Treg cells. Levels of iNOS, IL-12, IL-10, TGF-ß, Arg-1, and CCL-22 were measured using enzyme-linked immunosorbent assay. Co-IP was used to verify whether YAP binds to STAT3. Hematoxylin-eosin staining was used to observe tumor morphology. Cell counting kit-8 was selected to detect T-cell proliferation. RESULTS: YAP, STAT3, P-STAT3, VEGF, VEGFR-2, and PD-L1 were highly expressed in BC tissues. The M2/M1 macrophages ratio increased in the TAMs group compared with the control group. Inhibiting of YAP and STAT3 decreased the M2/M1 macrophages ratio. YAP was found to bind with STAT3. T-cell proliferation was enhanced after YAP inhibition, and overexpression of STAT3 reversed the regulation of YAP on T-cell proliferation. In animal studies, inhibiting YAP inhibited tumor weight and volume development. After YAP inhibition, inflammatory infiltration, M2/M1 macrophage ratio, and Treg cell ratio declined, while CD8+ and CD4+ T-cell ratio increased. CONCLUSION: In conclusion, this study suggested inhibition of YAP/STAT3 reversed M2 polarization of TAMs and suppressed CD8+ T-cell activity in the BC immune microenvironment. These findings open up new avenues for the development of innovative therapies in the treatment of BC.

The Mechanism of Houttuynia cordata Embryotoxicity Was Explored in Combination with an Experimental Model and Network Pharmacology.

Houttuynia cordata (H. cordata) is the most common herb as a food and traditional Chinese medicine. Currently, studies on its toxicity have mainly focused on hepatotoxicity. However, its potential embryotoxicity by long-term exposure is often overlooked. Objective: To investigate the effects of H. cordata on embryonic development and its toxicity mechanism by combining network pharmacology, molecular docking, and in vitro experimental methods. Methods: The effects of H. cordata on embryos were evaluated. Zebrafish embryos and embryoid bodies were administered to observe the effects of H. cordata on embryonic development. Based on network pharmacological analysis, it was found that the main active agents producing toxicity in H. cordata were oleanolic acid, lignan, and aristolactam AII. H. cordata can affect PI3K-Akt, MAPK, and Ras signaling pathways by regulating targets, such as AKT1, EGFR, CASP3, and IGF-1. RT-PCR and immunohistochemistry results showed that the expression of AKT1 and PI3K in the embryoid body was significantly reduced after drug administration (p < 0.05). Conclusions: The results of network pharmacology and in vitro experiments suggest that H. cordata may affect embryonic development by influencing the PI3K-Akt signaling pathway.

Osteosarcoma transcriptome data exploration reveals STC2 as a novel risk indicator in disease progression.

BACKGROUND: Osteosarcoma has been the most common primary bone malignant tumor in children and adolescents. Despite the considerable improvement in the understanding of genetic events attributing to the rapid development of molecular pathology, the current information is still lacking, partly due to the comprehensive and highly heterogeneous nature of osteosarcoma. The study is to identify more potential responsible genes during the development of osteosarcoma, thus identifying promising gene indicators and aiding more precise interpretation of the disease. METHODS: Firstly, from GEO database, osteosarcoma transcriptome microarrays were used to screen the differential expression genes (DEGS) in cancer comparing to normal bone samples, followed by GO/KEGG interpretation, risk score assessment and survival analysis of the genes, for the purpose of selecting a credible key gene. Further, the basic physicochemical properties, predicted cellular location, gene expression in human cancers, the association with clinical pathological features and potential signaling pathways involved in the key gene's regulation on osteosarcoma development were in succession explored. RESULTS: Based on the selected GEO osteosarcoma expression profiles, we identified the differential expression genes in osteosarcoma versus normal bone samples, and the genes were classified into four groups based on the difference level, further genes interpretation indicated that the high differently level (> 8 fold) genes were mainly located extracellular and related to matrix structural constituent regulation. Meanwhile, module function analysis of the 67 high differential level (> 8 fold) DEGS revealed a 22-gene containing extracellular matrix regulation associated hub gene cluster. Further survival analysis of the 22 genes revealed that STC2 was an independent prognosis indicator in osteosarcoma. Moreover, after validating the differential expression of STC2 in cancer vs. normal tissues using local hospital osteosarcoma samples by IHC and qRT-PCR experiment, the gene's physicochemical property revealed STC2 as a cellular stable and hydrophilic protein, and the gene's association with osteosarcoma clinical pathological parameters, expression in pan-cancers and the probable biological functions and signaling pathways it involved were explored. CONCLUSION: Using multiple bioinformatic analysis and local hospital samples validation, we revealed the gain of expression of STC2 in osteosarcoma, which associated statistical significantly with patients survival, and the gene's clinical features and potential biological functions were also explored. Although the results shall provide inspiring insights into further understanding of the disease, further experiments and detailed rigorous clinical trials are needed to reveal its potential drug-target role in clinical medical use.

Molecular profiling of gene fusions in soft tissue sarcomas by Ion AmpliSeqTM: a study of 35 cases.

Background: The accurate diagnosis of sarcoma can be difficult as there are over 70 different subtypes. While molecular profiling in soft tissue sarcoma (STS) has gradually been incorporated into routine diagnostics, conventional methods such as fluorescence in situ hybridization (FISH), reverse transcriptase-PCR (RT-PCR), and Sanger sequencing have several drawbacks. By allowing simultaneous analysis of multiple targets and increasing sequencing depth to achieve ultra-sensitivity, next-generation sequencing (NGS) can not only detect common genetic abnormalities without prior assumptions but also identify uncommon or even new variants. Methods: In this study, the applicability of NGS in assessing STS using the Ion Torrent Proton was evaluated and compared with other methods. A cohort of 35 tissue specimens from STS patients, including alveolar soft-part sarcoma (ASPS), Ewing's sarcoma (ES), synovial sarcoma (SS), dermatofibrosarcoma protuberans (DFSP), and myxoid liposarcoma (MLPS) patients, were subjected to NGS by an Ion AmpliSeqTM Custom panel. Results: A proportion of 97.14% (34/35) were successfully conducted to detect gene fusion positive events and met all criteria for good quality. The concordance between NGS and conventional techniques was 94.12% (32/34). NGS also showed superior results, as Sanger sequencing and FISH in two cases were false negatives, demonstrating the excellent diagnostic utility of NGS for translocation detection in STS. Conclusions: The results in this study show the potential for NGS to aid in diagnosis and clinical monitoring of STS and warrant additional studies in larger cohorts.

Twinfilin regulates actin assembly and Hexagonal peroxisome 1 (Hex1) localization in the pathogenesis of rice blast fungus Magnaporthe oryzae.

Actin assembly at the hyphal tip is key for polar growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. The mechanism of its precise assemblies and biological functions is not understood. Here, we characterized the role of M. oryzae Twinfilin (MoTwf) in M. oryzae infection through organizing the actin cables that connect to Spitzenkörper (Spk) at the hyphal tip. MoTwf could bind and bundle the actin filaments. It formed a complex with Myosin2 (MoMyo2) and the Woronin body protein Hexagonal peroxisome 1 (MoHex1). Enrichment of MoMyo2 and MoHex1 in the hyphal apical region was disrupted in a ΔMotwf loss-of-function mutant, which also showed a decrease in the number and width of actin cables. These findings indicate that MoTwf participates in the virulence of M. oryzae by organizing Spk-connected actin filaments and regulating MoHex1 distribution at the hyphal tip.