GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection.

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.

Integrating Metabolomics and Network Pharmacology to Explore the Mechanism of Tongmai Yangxin Pills in Ameliorating Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX) is a broad-spectrum chemotherapeutic drug used in clinical treatment of malignant tumors. It has a high anticancer activity but also high cardiotoxicity. The aim of this study was to explore the mechanism of Tongmai Yangxin pills (TMYXPs) in ameliorating DOX-induced cardiotoxicity through integrated metabolomics and network pharmacology. In this study, first, an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) metabonomics strategy was established to obtain metabolite information and potential biomarkers were determined after data processing. Second, network pharmacological analysis was used to evaluate the active components, drug-disease targets, and key pathways of TMYXPs to alleviate DOX-induced cardiotoxicity. Targets from the network pharmacology analysis and metabolites from plasma metabolomics were jointly analyzed to select crucial metabolic pathways. Finally, the related proteins were verified by integrating the above results and the possible mechanism of TMYXPs to alleviate DOX-induced cardiotoxicity was studied. After metabolomics data processing, 17 different metabolites were screened, and it was found that TMYXPs played a role in myocardial protection mainly by affecting the tricarboxylic acid (TCA) cycle of myocardial cells. A total of 71 targets and 20 related pathways were screened out with network pharmacological analysis. Based on the combined analysis of 71 targets and different metabolites, TMYXPs probably played a role in myocardial protection through regulating upstream proteins of the insulin signaling pathway, MAPK signaling pathway, and p53 signaling pathway, as well as the regulation of metabolites related to energy metabolism. They then further affected the downstream Bax/Bcl-2-Cyt c-caspase-9 axis, inhibiting the myocardial cell apoptosis signaling pathway. The results of this study may contribute to the clinical application of TMYXPs in DOX-induced cardiotoxicity.

Identification and Functional Analysis of SabHLHs in Santalum album L.

Santalum album L., a semi-parasitic evergreen tree, contains economically important essential oil, rich in sesquiterpenoids, such as (Z) α- and (Z) ß-santalol. However, their transcriptional regulations are not clear. Several studies of other plants have shown that basic-helix-loop-helix (bHLH) transcription factors (TFs) were involved in participating in the biosynthesis of sesquiterpene synthase genes. Herein, bHLH TF genes with similar expression patterns and high expression levels were screened by co-expression analysis, and their full-length ORFs were obtained. These bHLH TFs were named SaMYC1, SaMYC3, SaMYC4, SaMYC5, SabHLH1, SabHLH2, SabHLH3, and SabHLH4. All eight TFs had highly conserved bHLH domains and SaMYC1, SaMYC3, SaMYC4, and SaMYC5, also had highly conserved MYC domains. It was indicated that the eight genes belonged to six subfamilies of the bHLH TF family. Among them, SaMYC1 was found in both the nucleus and the cytoplasm, while SaMYC4 was only localized in the cytoplasm and the remaining six TFs were localized in nucleus. In a yeast one-hybrid experiment, we constructed decoy vectors pAbAi-SSy1G-box, pAbAi-CYP2G-box, pAbAi-CYP3G-box, and pAbAi-CYP4G-box, which had been transformed into yeast. We also constructed pGADT7-SaMYC1 and pGADT7-SabHLH1 capture vectors and transformed them into bait strains. Our results showed that SaMYC1 could bind to the G-box of SaSSy, and the SaCYP736A167 promoter, which SaSSy proved has acted as a key enzyme in the synthesis of santalol sesquiterpenes and SaCYP450 catalyzed the ligation of santalol sesquiterpenes into terpene. We have also constructed pGreenII 62-SK-SaMYC1, pGreenII 0800-LUC-SaSSy and pGreenII 0800-LUC-SaCYP736A167 via dual-luciferase fusion expression vectors and transformed them into Nicotiana benthamiana using an Agrobacterium-mediated method. The results showed that SaMYC1 was successfully combined with SaSSy or SaCYP736A167 promoter and the LUC/REN value was 1.85- or 1.55-fold higher, respectively, than that of the control group. Therefore, we inferred that SaMYC1 could activate both SaSSy and SaCYP736A167 promoters.

The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma.

BACKGROUND: A-kinase interacting protein 1 (AKIP1) is recently implicated in the pathogenesis of several solid tumors, while its role in glioblastoma multiforme (GBM) is largely unknown. Therefore, the current study aimed to investigate the effect of AKIP1 on GBM cell malignant behaviors, stemness, and its underlying molecular mechanisms. METHODS: U-87 MG and A172 cells were transfected with control or AKIP1 overexpression plasmid; control or AKIP1 siRNA plasmid. Then cell proliferation, apoptosis, invasion, CD133+ cell proportion, and sphere formation assays were performed. Furthermore, RNA-Seq was performed in U-87 MG cells. Besides, AKIP1 expression was detected in 25 GBM and 25 low-grade glioma (LGG) tumor samples. RESULTS: AKIP1 was increased in several GBM cell lines compared to the control cell line. After transfections, it was found that AKIP1 overexpression increased cell invasion, CD133+ cell proportion, and sphere formation ability while less affecting cell proliferation or cell apoptosis in U-87 MG and A172 cells. Moreover, AKIP1 siRNA achieved the opposite effect in these cells, except that it inhibited cell proliferation but induced cell apoptosis to some extent. Subsequent RNA-Seq assay showed several critical carcinogenetic pathways, such as PI3K/AKT, Notch, EGFR tyrosine kinase inhibitor resistance, Ras, ErbB, mTOR pathways, etc. were potentially related to the function of AKIP1 in U-87 MG cells. Clinically, AKIP1 expression was higher in GBM tissues than in LGG tissues, which was also correlated with the poor prognosis of GBM to some degree. CONCLUSIONS: AKIP1 regulates the malignant behaviors and stemness of GBM via regulating multiple carcinogenetic pathways.

Effector Sntf2 Interacted with Chloroplast-Related Protein Mdycf39 Promoting the Colonization of Colletotrichum gloeosporioides in Apple Leaf.

Glomerella leaf spot of apple, caused by Colletotrichumgloeosporioides, is a devastating disease that leads to severe defoliation and fruit spots. The Colletotrichum species secretes a series of effectors to manipulate the host's immune response, facilitating its colonization in plants. However, the mechanism by which the effector of C. gloeosporioides inhibits the defenses of the host remains unclear. In this study, we reported a novel effector Sntf2 of C. gloeosporioides. The transient expression of SNTF2 inhibits BAX-induced cell death in tobacco plants. Sntf2 suppresses plant defense responses by reducing callose deposition and H2O2 accumulation. SNTF2 is upregulated during infection, and its deletion reduces virulence to the plant. Sntf2 is localized to the chloroplasts and interacts with Mdycf39 (a chloroplast PSII assembly factor) in apple leaves. The Mdycf39 overexpression line increases susceptibility to C. gloeosporioides, whereas the Mdycf39 transgenic silent line does not grow normally with pale white leaves, indicating that Sntf2 disturbs plant defense responses and growth by targeting Mdycf39.

Shufeiya Recipe Improves Monocrotaline-Induced Pulmonary Hypertension in Rats by Regulating SIRT3/FOXO3a and Its Downstream Signaling Pathways.

Pulmonary hypertension (PH) is a chronic and progressive disease caused by obstructions and functional changes of small pulmonary arteries. Current treatment options of PH are costly with patients needing long-term taking medicine. The traditional Chinese medicine (TCM) compound "Shufeiya Recipe" was used to intervene in monocrotaline- (MCT-) induced pulmonary hypertension in rats. The rats were randomly divided into the control group, model group, positive drug (Sildenafil) group, and Shufeiya Recipe low-, moderate-, and high-dose groups. The improvement effect of the Shufeiya Recipe on the mean pulmonary artery pressure (mPAP) was assessed in PH rats, and pathological staining was used to observe the pathological changes of lung tissue. The impact of the Shufeiya Recipe on oxidative stress damage in rats with pulmonary hypertension and the regulation of SIRT3/FOXO3a and its downstream signaling pathways were determined. The results showed that Shufeiya Recipe could significantly downregulate mPAP and improve lung histopathological changes; downregulate serum levels of reactive oxygen species (ROS); upregulate the concentrations of COX-1 and COX-2 and the activity of Mn-SOD; inhibit oxidative response damage; promote the protein expression of SIRT3, FOXO3a, p-PI3K, p-AKT, and p-eNOS; increase the level of expression of NO, sGC, cGMP, and PKG; and downregulate the level of protein expression of Ras, p-MEK1/2, p-ERK1/2 and c-fos. These results indicate that Shufeiya Recipe can improve MCT-induced pulmonary hypertension in rats by regulating SIRT3/FOXO3a and its downstream PI3K/AKT/eNOS and Ras/ERK signaling pathways.

Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album.

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Lentinan combined with cisplatin for the treatment of non-small cell lung cancer.

BACKGROUND: A growing number of studies suggest that lentinan combined with cisplatin thoracic injection for the treatment of lung cancer is an effective combination of traditional Chinese and Western medicine, which has a continuous and beneficial effect on eliminating clinical symptoms and improving cachexia in lung cancer patients. However, whether this treatment is effective and safe for lung cancer patients or not, evidence supporting the effectiveness and safety of this treatment is still incomplete. Besides, there is lack of systematic review to assess the detailed situation (including risk of bias and methodology) of current related clinical studies. OBJECTIVE: This study aimed to evaluate the effectiveness and safety of lentinan combined with cisplatin thoracic injection in the treatment of lung cancer. METHODS: The major databases (Embase, PubMed, the Cochrane Library, China National Knowledge Infrastructure, Chinese Scientific Journals Database [VIP] Database, Chinese Biomedical Literature Service System [SinoMed], and Wanfang Database) were searched from inception to March 1, 2020. Randomized controlled trials (RCTs) of lentinan combined with cisplatin chest injection on patients with non-small cell lung cancer (NSCLC) were identified. Two assessors reviewed each trial independently. The methodological quality of the eligible studies was evaluated according to the Cochrane Collaboration's tool for assessing risk of bias. Both the data extraction and the literature quality screening evaluation were conducted independently by 2 researchers. RESULTS: Totally 17 clinical RCTs were included in this study, involving 1390 patients. Meta-analysis results showed that the clinical efficacy (risk ratio [RR] = 1.34, 95% confidence interval [CI] 1.21-1.48), effective subgroup analysis (RR = 1.51, 95% CI 1.3-1.77), and quality of life (RR = 1.48, 95% CI 1.27-1.72), the differences are statistically significant. In terms of adverse reactions, mainly related to gastrointestinal reactions and bone marrow suppression, the incidence and degree of adverse reactions of lentinan combined with cisplatin thoracic injection group were lower than those of cisplatin thoracic injection group alone. CONCLUSIONS: The current evidence prompted that Lentinan combined with cisplatin in thoracic injection might benefit patients with NSCLC on a certain extent; this systematic review revealed some definite conclusions about the application of Lentinan combined with cisplatin in thoracic injection for NSCLC. Due to the low methodological quality, high risk of bias, and inadequate reporting on clinical data, these results still require verification by a large number of well designed, heterogeneous RCT studies. More rigorous, multicenter, sufficient-sample, and double-blind RCTs are warranted.

A Network Pharmacology-Based Approach to Investigating the Mechanisms of Fushen Granule Effects on Intestinal Barrier Injury in Chronic Renal Failure.

PURPOSE: Fushen Granule (FSG) is a Chinese medicine prepared by doctors for treating patients with chronic renal failure, which is usually accompanied by gastrointestinal dysfunction. Here, we explore the protective effect of FSG on intestinal barrier injury in chronic renal failure through bioinformatic analysis and experimental verification. METHODS: In this study, information on the components and targets of FSG related to CRF is collected to construct and visualize protein-protein interaction networks and drug-compound-target networks using network pharmacological methods. DAVID is used to conduct gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, it is validated by in vitro experiments. In this study, the human intestinal epithelial (T84) cells are used and divided into four groups: control group, model group, FSG low-dose group, and FSG high-dose group. After the experiment, the activity of T84 cells is detected by a MTT assay, and the expressions of tight junction protein ZO-1, claudin-1, nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), malondialdehyde (MDA), and cyclooxygenase-2 (COX-2) are examined by immunofluorescence and/or western blotting. RESULTS: Eighty-six potential chronic renal failure-related targets are identified by FSG; among them, nine core genes are screened. Furthermore, GO enrichment analysis shows that the cancer-related signaling pathway, the PI3K-Akt signaling pathway, the HIF1 signaling pathway, and the TNF signaling pathway may play key roles in the treatment of CRF by FSG. The MTT method showed that FSG is not cytotoxic to uremic toxin-induced injured T84 cells. The results of immunofluorescence and WB indicate that compared with the control group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is decreased and protein expressions level of HO-1, MDA, and COX-2 is increased after urinary toxin treatment. Instead, compared with the model group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is increased and protein expressions level of HO-1, MDA, and COX-2 is decreased after FSG treatment. CONCLUSION: FSG had a protective effect on urinary toxin-induced intestinal epithelial barrier injury in chronic renal failure, and its mechanism may be related to the upregulation of Nrf2/HO-1 signal transduction and the inhibition of tissue oxidative stress and inflammatory responses. Screening CRF targets and identifying the corresponding FSG components by network pharmacological methods is a practical strategy to explain the mechanism of FSG in improving gastrointestinal dysfunction in CRF.

Identification of biomarkers and construction of a microRNA­mRNA regulatory network for clear cell renal cell carcinoma using integrated bioinformatics analysis.

With the recent research development, the importance of microRNAs (miRNAs) in renal clear cell carcinoma (CCRCC) has become widely known. The purpose of this study is to screen out the potential biomarkers of renal clear cell carcinoma (CCRCC) by microarray analysis. The miRNA chip (GSE16441) and mRNA chip (GSE66270) related to CCRCC were downloaded from the Gene Expression Omnibus (GEO) database. After data filtering and pretreating, R platform and a series of analysis tools (funrich3.1.3, string, Cytoscape_ 3.2.1, David, etc.) were used to analyze chip data and identify the specific and highly sensitive biomarkers. Finally, by constructing the miRNA -mRNA interaction network, it was determined that five miRNAs (hsa-mir-199a-5p, hsa-mir-199b-5p, hsa-mir-532-3p and hsa-mir-429) and two key genes (ETS1 and hapln1) are significantly related to the overall survival rate of patients.

[Study on different ploidy Siraitia grosvenorii and their genetic relationship by FTIR].

Siraitia grosvenorii was an important Chinese traditional medicine. The spectra of leaf of diploid, triploid and tetraploid Siraitia grosvenorii were determined by Fourier transform infrared spectroscopy (FTIR) with OMNI sampler directly, fast and accurately. And based on the indices of wave number-absorbance from different bands and through comparison of differences of these infrared spectra the ploidy difference and genetic relationship of Siraitia grosvenorii were studied by the methods of principal component analysis (PCA) and cluster analysis. The results showed that for the ploidy, the position relationship of PCA three-dimensional-plot and the distance coefficient of clustering analysis plot between diploid and tetraploid were most remarkbly different, and the triploid was basically between diploid and tetraploid, so the ploidy was more different, the position relationship of PCA and the distance coefficient of clustering analysis were further, and the law was: 2 X < 3 X < 4 X. At the same time, the genetic relationship was further with each other while the position relationship of PCA and distance coefficient of clustering analysis was further too. And the genetic relationship of triploid was affected by the different male parent while their female parent was the same one. Therefore, using FTIR based on PCA and cluster analysis we could reveal the difference of ploidy and the genetic relationship between Siraitia grosvenorii to a certain extent. FTIR could be used for excellently polyploidy species breeding of Siraitia grosvenorii.

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl.

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa. Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.