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Which is the key?????

Which is the key?????. Any biological process is controlled by chemical signals. By knowing of these signal we control any biological process in any organism. Can we talk with microorganisms? Can we give them instructions?. Can we talk with plants? Can we give them instructions?.

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Which is the key?????

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  1. Which is the key????? Any biological process is controlled by chemical signals. By knowing of these signal we control any biological process in any organism.

  2. Can we talk with microorganisms? Can we give them instructions?

  3. Can we talk with plants? Can we give them instructions?

  4. Plants and microbial signals discovery By Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology alrahman3@hotmail.com Tel: 002-01228188759

  5. Any biological process is controlled by chemical signals. • By knowing these chemical signals we can control, talk or give instructions to plants, microorganisms or insects.

  6. Synergism Biosurfactants Antagonism Microbial signals Enzymes and proteins Quorum sensing Siderophores Microbial products

  7. This image from: http://2011.igem.org/Team:Grenoble/Projet/Modelling/Deterministic Slime molds plasmodium formation is controlled by c-AMP. c-AMP Quorum sensing control of P. aeruginosa Nikki Moran, http://blogs.rsc.org/mb/2011/01/25/lyngbyoic-acid-disrupts-quorum-sensing-in-p-aeruginosa/

  8. Synergism Plant products Antagonism Plant signals Enzymes and proteins Root exudates Pests activators Pests inhibitors

  9. LRR proteins Pectinases and cellulases inhibiting proteins belong to the leucine-rich repeat (LRR) family and are known to prevent fungal pathogen invasion. Phytoalexins Strigolatcgones Faba bean-Orobanche seeds germination stimulation A crucifer plant infected with S. sclerotiorum From the graphical abstract of M. Soledade C. Pedras and Mohammad Hossain,  Org. Biomol. Chem., 2006, 4, 2581

  10. Stages for a new drug discovery 1- The drug source selection 2- Extraction and purification 3- Biological activity determination 4- Chemical structure elucidation

  11. So, The processes of new drug discovery requires co-work and expertise of a wide variety of scientific, technical and managerial groups

  12. 2- Extraction Proteineous compounds (From plant tissues, root exudates, microbial broth or cells) Non- proteineous compounds (From plant tissues, root exudates, microbial broth or cells) 1- The drug source Plant (Plant tissues or root exudates) Microorganisms Bacteria, fungi, actinomycetes, algae 3- Testing of each obtained extract for a biological activity (Against bacteria, fungi, parasitic weeds, insects, nematodes, animal disease,……)

  13. 4- Purification of the active compound from the obtained extract Purification of proteineous compounds: - Concentration (precipitation) - Fractionation (gel filtration, ion exchanger). - Electrophoresis (two-dimensional). Purification of non-proteineous compounds: - Liquid-liquid extraction. - Liquid- solid extraction. - Chromatographic methods (normal phase and reversed phase). - HPLC-MS analysis 5- Chemical structure elucidation • Proteineous compounds: • - MW determination. • - Subunits determination. • - Amino acids sequencing. • Crystallization. • NMR ( 2D and 3D). non-proteineous compounds: - NMR analysis (one and two dimensions - IR analysis - Mass spectroscopy - X-ray crystallography

  14. 1- The drug sources: 1) Plants: Several plants contain pharmaceutical compounds, pesticides, ….. Several plant extracts or root exudates showed a biological activity but the active compound is still not identified. You can found a grass grow on the train run-way contains a new antimicrobial or medicinal compound.

  15. Root exudates collection: 1- Cultivate the plant in pots and irrigate with nutrient solution for a period vary with the plant type. 2- Incubate in a glasshouse or incubation chamber under the optimum growth conditions. 3- remove the plant carefully from the soil and wash the root by tap water. 4- Soak in a conical flask containing distilled water for one day only.

  16. 2) Microorganisms: Isolation of microorganisms: Serial dilution method I the best. The used medium is: 1- PDA for fungi 2- Nutrient agar for bacteria. 3- Casein starch agar for actinomycetes.

  17. 2- Testing for a biological activity: 1- Against a pathogen Against hydrolytic enzymes of the pathogen Against the pathogen itself Fungi Insect Pectinases Hemicell-ulases Nematodes Cellulases Bacteria Keratinase Parasitic weeds Other

  18. Inhibition tests against the pathogen itself By the plant extract or microbial supernatant Dual culture method 1- Inhibition test in solid media 2- Inhibition test in liquid media Control Medium + an extract

  19. 2- Tests against parasitic weeds (Germination inhibition or activation) Dual culture method By the extract . . . Magenta box Sterilized nutrient solution Tissue culture method Parasitic weeds seeds Glass plate covered by filter paper Control (Without the inhibitor or the activator)

  20. For germination stimulation: The compound is added in the GFFR or in the tissue culture medium compared with two control treatments, why? • For germination inhibition: - A stimulator (such as strigol) is firstly added on two GFFR disks. The tested inhibitor is then added on one of them. - The same test can be performed by tissue culture.

  21. 3- Inhibition test against insects: The plant extract, microbial broth or a suspension of the isolated microorganism is mixed with the nutrient materials of the tested insect. The life and dead insects are calculated after a few minutes, hours to days (according to the insect type) and compared with control.

  22. B- Inhibition test against a hydrolytic enzyme of a plant or human pathogen: This is performed through three steps: 1- Production of the hydrolytic enzyme (from bacteria, fungi or a parasitic weed). 2- Determination of the enzyme activity in presence and absence of the tested substance.

  23. 3- Extraction of the active drug The nature of the active drug can be: 1- Protein. 2- Non-proteineous. 3- Complex: Glycoside, glycolipid, lipoprotein, glycoprotein,……

  24. a) Extraction of the proteineous compounds: - Selection of the extraction buffer. - Some additives are required during extraction b) Extraction of the non-proteineous compounds: - Selection of the extraction solvent. - The extraction method: Liquid- liquid extraction Liquid solid extraction Solid phase extraction (SPE) Super Critical Fluid instrument (SCF)

  25. 1) Liquid- liquid extraction techniques: 1- Shake flasks. 2- Separatory funnel

  26. 2) Solid-phase extraction (SPE) techniques: • The compounds in a mixture are separated according to their physical or chemical properties. • Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrix, including urine, blood, water, beverages, soil, animal tissue, plant extracts or microbial supernatants.

  27. Types of solid phases in SPE: • Reversed phase (C8 or C18) • Normal phase (Silica gel or CN) • Ion exchanger (Cation or anion exchangers) • Affinity • Gel filtration.

  28. 3) Supercritical Fluid Extraction • Supercritical Fluid Extraction (SCF or SFE) is the process of separating one component from a sample usingsupercritical fluidsas the extractingsolvent. • Carbon dioxide (CO2is the most used supercritical fluid, sometimes modified by co-solvents such asethanolormethanol

  29. Purity determination • Each of the obtained extracts must be tested for the biological activity. • The active extract is then analyzed for its purity determination: 1- Uv-Vis scanning is the first analysis to identify λ-max.

  30. 2- The HPLC is used by testing two columns: reversed phase and normal phase, and the detection wave length that was obtained from the scanning. 3- The HPLC-MS is performed utilizing the previous HPLC data. The HPLC tells us how many compounds in the active extract.

  31. 4- Purification • The purification is the most important step. The purification is performed by: • TLC • Glass column • Preparative HPLC

  32. The purity must be determined by HPLC/MS and the activity of final pure compound must be determined before chemical structure elucidation. HPLC chromatogram showed one pure compound

  33. 5- Chemical structure elucidation This is performed by spectroscopy techniques: 1- IR spectrophotometer: For identification of the functional groups. 2- Mass spectrophotometer: For determination of MW, base ion and mass fragmentation pattern. 3- One and two dimensions NMR: H1, C13, COSY, NOESY, HMABC, HMQC, APT, DEPT. 4- Crystallization and analysis by X-ray single crystal.

  34. 1) IR analysis It identifies the function groups.

  35. 2) Mass spectrometer: Identifies the MW and fragmentation pattern

  36. Mass spectrometry of organic compounds

  37. 3) NMR analysis: One dimension: H1 C13 APT DEPT 45 DEPT 90 DEPT 135 Two dimensions: COSY NOESY TOCSY HMBC HMQC HSQC

  38. H1-NMR Identifies number and types of hydrogen ions. Data interpretation: 1- Chemical shift values. 2- signals splitting. 3- Signals area. 4- j values

  39. C13 NMR Identifies the number and type of carbon atoms. Types of C13 NMR analysis: 1- 1D C13 NMR 2- DEPT 3- APT C13 data interpretation

  40. C13 NMR spectrum of acetophenone

  41. APT The carbon attached with odd number of protons showed signals up. The carbon attached with even number of protons showed signals down.

  42. Distortionless Enhancement by Polarization Transfer (DEPT) DEPT 45 DEPT 135 DEPT 90

  43. Correlation Spectroscopy (COSY) • Correlation between the proton with the adjacent proton (Three bonds correlation), (H-H correlation).

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