Integration of Transcriptomic and Proteomic Data Reveals the Possible Action Mechanism of the Antimicrobial Zhongshengmycin against Didymella segeticola, the Causal Agent of Tea Leaf Spot

2021 ◽  
Author(s):  
Yafeng Ren ◽  
Dongxue Li ◽  
Shilong Jiang ◽  
Yu Wang ◽  
Qin Tang ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Mode Of Action ◽  
Morphological Changes ◽  
Extracellular Polysaccharides ◽  
Proteomics Data ◽  
Protein Levels ◽  
Proteomic Data ◽  
Half Maximal Effective Concentration ◽  
Tea Leaf

Tea leaf spot, caused by the fungal phytopathogen Didymella segeticola (Q. Chen) Q. Chen, Crous & L. Cai (syn. Phoma segeticola, is an important foliar disease, which can cause a huge loss in terms of the production and quality of tea for no effective management measures to control the disease. This study screened a natural antimicrobial chemical for its activity against D. segeticola and studied its mode of action. Antifungal activity of the Streptomyces-derived antimicrobial Zhongshengmycin (ZSM) against D. segeticola strain GZSQ-4 was assayed in vitro by the mycelial growth rate method. Optical microscopy, scanning and transmission electron microscopy were used to observe the morphological effects on hyphae treated with ZSM, with these studies being complemented by transcriptomics, proteomics, and bioinformatics studies to identify the differentially expressed genes (DEGs) or the differentially expression proteins (DEPs) in hyphae treated with ZSM. Correlation analysis of transcriptomics and proteomics data were used to reveal the mode of action. The results indicated that ZSM could inhibit the growth of hyphae in vitro with a half-maximal effective concentration (EC50) of 5.9 μg mL-1, inducing some morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation, affecting the biosynthesis of some hyphal proteins at the mRNA and protein levels, and revealing correlation between findings from transcriptomes and proteomes.

The Antifungal Activity and Mode of Action of a Streptomyces-Derived Anti-Microbial, Ningnanmycin, on the Tea Gray Blight Disease Pathogen Pseudopestalotiopsis camelliae-sinensis

2021 ◽  
Author(s):  
Rui Yang ◽  
Shilong Jiang ◽  
Xiaodong Wen ◽  
Xingchen Song ◽  
Xue Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Antifungal Activity ◽  
Mode Of Action ◽  
Morphological Changes ◽  
Response To Treatment ◽  
Effective Control ◽  
Extracellular Polysaccharides ◽  
Serious Disease ◽  
Blight Disease

Gray blight disease is a serious disease of tea (Camellia sinensis (L.) Kuntz), for which there is currently no effective control or preventative measure apart from fungicides. Screening for effectiveness of a natural anti-microbial against this pathogen and identifying its mode of action could contribute to the management of this disease. Antifungal activity of the anti-microbial Ningnanmycin (NNM) from Streptomyces noursei var. xichangensis against the pathogen causing gray blight disease, Pseudopestalotiopsis camelliae-sinensis (F. Liu & L. Cai) strain GZHS-2017-010 was confirmed in vitro by the mycelial growth rate method. Optical microscopy, scanning electron microscopy and transmission electron microscopy were used to observe morphological change of hyphae of Ps. camelliae-sinensis treated with NNM. RNA sequencing, bioinformatics and quantitative real-time PCR were used to identify genes in the hyphae which were differentially expressed in response to treatment with NNM. Thirty-eight genes from sixteen pathways, known as targets of antifungal agents, were used to investigate gene expression in hyphae at the half-maximal effective concentration (EC50) dosage, EC30, and EC70 for 1, 7, or 14 h. The results indicated that NNM can inhibit the growth of hyphae in vitro, with an EC50 of 75.92 U/mL, inducing morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation in protein synthesis, and influencing some biosynthetic functions of the hyphae.

scholarly journals The antifungal mechanism of the biogenic antimicrobial Ningnanmycin on Didymella segeticola involves binding to tryptophanyl-tRNA synthetase, inhibiting translation

2020 ◽  
Author(s):  
Li Dongxue ◽  
Qiaoxiu Yin ◽  
Xue Wang ◽  
Shilong Jiang ◽  
Dissanayake Saman Pradeep Dharmasena ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Mycelial Growth ◽  
Morphological Changes ◽  
Binding Free Energy ◽  
Trna Synthetase ◽  
Guizhou Province ◽  
Leaf Spot Disease ◽  
Mrna Levels ◽  
Action Mechanism ◽  
Tea Leaf

Abstract BackgroundTea [Camellia sinensis (L.) Kuntze] has been recently cultivated in Guizhou Province, China, where the cultivated area has reached 350,000 hectares, making it the major tea-growing region in world. Tea leaf spot caused by Didymella segeticola can induce the decreases in quality and quantity of tea leaves, which is an important disease in tea plantations at higher altitude, where cold spells occur in late spring. As a promising biogenic antimicrobial agent against crop diseases, Ningnanmycin (NNM) was produced from Streptomyces noursei var. xichangensisn, represented higher field efficiency against fungal, bacterial and viral phytopathogens, lower toxicity and lower residue. However, the action mechanism of NNM against phytopathogens just stays on the stage of anti-viral mechanism, which limits the application of NNM in the management of plant fungal diseases. Here, we studied the action mechanism of NNM against D. segeticola using many methods of transcriptome, ultrastructure, molecular biology and molecular docking.ResultsNNM strongly inhibited the mycelial growth of D. segeticola with the half-maximal effective concentration of 1287.54 U/mL. Optical, fluorescence, scanning and transmission electron microscopy were applied to observe morphological changes of cellular, organelle for D. segeticola treated by NNM. A great number of morphological changes of D. segeticola indicated that NNM could affect the biosynthesis of the phytopathogen. For further, RNA-Seq results showed that NNM treated D. segeticola induced 1,363 significantly differentially expressed genes (DEGs) comparing with the control (P < 0.05). The DEGs were highly enriched in structural component of ribosome, ribosome and translation by Gene Ontology, as well as in ribosome pathway at Kyoto Encyclopedia of Genes and Genomes. NNM regulated the mRNA levels of RPS7, RPS9, RPS10b, RPL9, RPL11 and TrpRS, and represent the different regulation mode by the comparative analysis with a classical translation extension inhibitor, cycloheximide. The molecular docking indicated that NNM possessed a marked affinity with TrpRS, with the binding free energy is -101.55 kcal/mol.ConclusionsNNM could potentially affect translation by binding to tryptophanyl-tRNA synthetase, thus inhibiting mycelial growth. This study will provide insights for anti-fungal mechanism of NNM and contribute to the control and prevention of tea leaf spot disease.

scholarly journals Integrated mRNA and small RNA sequencing for analyzing tea leaf spot pathogen Lasiodiplodia theobromae, under in vitro conditions and the course of infection

2020 ◽  
Author(s):  
Shilong Jiang ◽  
Qiaoxiu Yin ◽  
Dongxue Li ◽  
Xian Wu ◽  
Yong Wang ◽  
...  
Keyword(s):  
Small Rna ◽  
Leaf Spot ◽  
Guizhou Province ◽  
Phytopathogenic Fungus ◽  
Future Research ◽  
Small Rna Sequencing ◽  
Rna Sequences ◽  
Lasiodiplodia Theobromae ◽  
Tea Leaf

Lasiodiplodia theobromae (Pat.) Griffon & Maubl. is a phytopathogenic fungus, which can cause many different diseases on different crops. The pathogen can cause leaf spot on tea plants (Camellia sinensis), which negatively affects the productivity and quality of tea leaves in tea plantations in Guizhou Province, China. Although the genome sequence of L. theobromae has been published, no data on the transcriptome or small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf are available. Here, we report the high-quality transcriptome and small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf using the platform of Illumina HiSeq. This comprehensive expression profiling of the fungal pathogen will provide a valuable resource for future research on trait-specific genes of the pathogen, host-pathogen interactions and on disease resistance in the host.

Apigenin induces apoptosis in human lung cancer H460 cells through caspase- and mitochondria-dependent pathways

2010 ◽  
Vol 30 (8) ◽  
pp. 1053-1061 ◽  
Author(s):  
Hsu-Feng Lu ◽  
Yu-Jie Chie ◽  
Ming-Sung Yang ◽  
Kung-Wen Lu ◽  
Jene-John Fu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Human Lung ◽  
Human Cancer ◽  
Morphological Changes ◽  
Human Lung Cancer ◽  
Induce Cell Cycle Arrest ◽  
Protein Levels ◽  
Apoptosis Protein ◽  
H460 Cells

Apigenin (4,5,7-trihydroxyflavone), a promising chemopreventive agent presented in fruits and vegetables, has been shown to induce cell cycle arrest and apoptosis in many types of human cancer cell lines. However, there is no available information to address the effects of apigenin on human lung cancer H460 cells. In the present studies, H460 cells were treated with apigenin for different time and then were analyzed for the morphological changes, induction of apoptosis, protein levels associated with apoptosis and results in dose-dependent induction of morphological changes, decrease in the percentage of viability, induced DNA damage and apoptosis; down-modulation of the protein expression of Bid, Bcl-2, procaspase-8; up-regulation of protein levels of Bax, caspase-3, AIF, cytochrome c, GRP78 and GADD153; decreased the levels of mitochondrial membrane potential and increased the productions of reactive oxygen species (ROS) and Ca2+ in H460 cells. Taken together, this is the first systematic in vitro study showing the involvement of apoptosis regulatory proteins as potential molecular targets of apigenin in human lung cancer H460 cells.

Studies on Platelet Storage Biomarkers: Effect of Different Protein Extraction Buffers on Platelet Gelsolin and B-Actin Profiling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4075-4075
Author(s):  
Sandhya Kulkarni ◽  
Meganathan Kannan ◽  
Chintamani D Atreya
Keyword(s):  
Protein Extraction ◽  
Morphological Changes ◽  
Surrogate Marker ◽  
Buffer System ◽  
Protein Levels ◽  
Platelet Protein ◽  
Platelet Storage ◽  
Platelet Proteins

Abstract Platelet concentrates (PCs) are one of the blood cell components that play a major role in transfusion medicine. The shelf-life of PC is 5–7 days at 22°C. During storage PCs undergo certain morphological changes as well as physiological changes such as loosing of its surface glycoproteins, and reduced aggregation response to its agonists, which are collectively referred to as “storage lesions”. To date there is no in vitro “gold standard” that unequivocally serves as a surrogate marker to predict PC quality in vivo following transfusion. A comprehensive survey of reports in this field clearly indicate that there is some confusion with regards to the determination of which platelet protein or a panel of proteins could serve as potential markers associated with PC storage. Following a careful review of all available information, we recognized that perhaps one core issue to the observed discrepancies could be the buffer system that is being utilized by individual laboratories in extracting proteins in profiling protein alterations in PCs during storage. To address this, in the present study we analyzed two platelet proteins, b-actin and gelsolin-related 40 kDa protein. Profiles of these two proteins were previously reported by others to be altered during PC storage and hence implicated to be useful as biomarkers of platelet storage. By employing 3 different protein extraction buffer systems to isolate proteins from platelets at three storage intervals (Table 1), our results indicate that b-actin and gelsolin 40 kDa protein levels do not appear to change with time in platelet storage and therefore may not be useful as platelet storage markers and Buffer systems do make a difference in extracting different platelet proteins for analysis, for example 40 kDa gelsolin was not extractable in buffer 3 (Table 1), suggesting that one should be careful in selecting a buffer system for platelet proteomics to have meaningful results. Same level of 40 kDa Gelsolin Same level of b-actin Buffer system Day 0 Day 5 Day 16 Day 0 Day 5 Day 16 Buffer 1 Not identified Yes Yes Yes Yes Yes Buffer 2 Not identified Yes Yes Yes Yes Yes Buffer 3 Not identified Not identified Not identified Yes Yes Yes The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

Effect of Temperature, Velocity Gradient and I.V. Heparin on in Vitro Blood Coagulation and Platelet Aggregation

1967 ◽  
Vol 17 (01/02) ◽  
pp. 112-119 ◽  
Author(s):  
L Dintenfass ◽  
M. C Rozenberg
Keyword(s):  
Blood Coagulation ◽  
Velocity Gradient ◽  
Elevated Temperatures ◽  
Morphological Changes ◽  
Effect Of Temperature ◽  
Clotting Time ◽  
Progressive Change ◽  
Aggregation Of Platelets ◽  
Microscopic Techniques

SummaryA study of blood coagulation was carried out by observing changes in the blood viscosity of blood coagulating in the cone-in-cone viscometer. The clots were investigated by microscopic techniques.Immediately after blood is obtained by venepuncture, viscosity of blood remains constant for a certain “latent” period. The duration of this period depends not only on the intrinsic properties of the blood sample, but also on temperature and rate of shear used during blood storage. An increase of temperature decreases the clotting time ; also, an increase in the rate of shear decreases the clotting time.It is confirmed that morphological changes take place in blood coagula as a function of the velocity gradient at which such coagulation takes place. There is a progressive change from the red clot to white thrombus as the rates of shear increase. Aggregation of platelets increases as the rate of shear increases.This pattern is maintained with changes of temperature, although aggregation of platelets appears to be increased at elevated temperatures.Intravenously added heparin affects the clotting time and the aggregation of platelets in in vitro coagulation.

Cytotoxic Effect of the Combination of Gemcitabine and Atorvastatin Loaded in Microemulsion on the HCT116 Colon Cancer Cells

2017 ◽  
Vol 9 (2) ◽  
Author(s):  
Mayson H. Alkhatib ◽  
Dalal Al-Saedi ◽  
Wadiah S. Backer
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Anticancer Drugs ◽  
Therapeutic Potential ◽  
Morphological Changes ◽  
In Vitro Study ◽  
Colon Cancer Cells ◽  
Apoptosis Detection ◽  
Hct116 Cells

The combination of anticancer drugs in nanoparticles has great potential as a promising strategy to maximize efficacies by eradicating resistant, reduce the dosage of the drug and minimize toxicities on the normal cells. Gemcitabine (GEM), a nucleoside analogue, and atorvastatin (ATV), a cholesterol lowering agent, have shown anticancer effect with some limitations. The objective of this in vitro study was to evaluate the antitumor activity of the combination therapy of GEM and ATVencapsulated in a microemulsion (ME) formulation in the HCT116 colon cancer cells. The cytotoxicity and efficacy of the formulation were assessed by the 3- (4,5dimethylthiazole-2-yl)-2,5-diphyneltetrazolium bromide (MTT) assay. The mechanism of cell death was examined by observing the morphological changes of treated cells under light microscope, identifying apoptosis by using the ApopNexin apoptosis detection kit, and viewing the morphological changes in the chromatin structure stained with 4′,6-diamidino-2-phenylindole (DAPI) under the inverted fluorescence microscope. It has been found that reducing the concentration of GEM loaded on ME (GEM-ME) from 5μM to 1.67μM by combining it with 3.33μM of ATV in a ME formulation (GEM/2ATV-ME) has preserved the strong cytotoxicity of GEM-ME against HCT116 cells. The current study proved that formulating GEM with ATV in ME has improved the therapeutic potential of GEM and ATV as anticancer drugs.

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