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The Effects of Synthetic Iron Chelators and Flavonoids on Siderophore-producing P. fluorescensKamila Zakowicz
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Big Picture
▪ Siderophore producing bacteria
▪ Iron chelation therapy to induce hypoferramia
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Background
▪ Iron is essential
▪ 25% of world population is affected by iron abnormalities [1]
▪ 4 main iron abnormalities
Iron deficiency
Iron overload
Chronic disease induced anemia
Free radical pathology
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Free Radical Pathogenicity
▪ Free radicals are highly reactive molecules with an unpaired electron that are essential for biological processes
▪ can cause cell damage when they bind to important cell structures
▪ much of the cell degeneration and cell aging in certain chronic diseases, such as kidney disease and Alzheimer’s, is attributed to free radical oxidative damage [2]
▪ play a role in numerous infectious diseases
Body induces hypoferremia in pathogen (iron deficiency)
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Hypoferramia
▪ To combat the free radicals in infectious diseases, the body uses two different pathways to induce hypoferremia (anaemia) in the infectious disease: the hepcidin-dependent pathway and an hepcidin-independent pathway
▪ hepcidin-dependent:
body will increase its production of hepcidin, a metabolic iron-regulating mechanism, to reduce iron plasma and induce hypoferremia of the infectious disease
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Hypoferremia
▪ Hepcidin independent:
iron chelation may be used in order to to correct an iron abnormality caused by an infectious disease
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Why P. fluorescens?
▪ Nonpathogenic but produces siderophores called pyoverdine
▪ Same siderophores as P. areuginosa
Fig. 1: P. fluorescens (Bio Control Agents. (n.d.).
Retrieved April 03, 2017, from
https://www.indiamart.com/rocky-imports-
exports/bio-control-agents.html)
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Siderophores
▪ Produced only by certain bacteria
▪ Mechanisms that help facilitate iron uptake in iron deficient conditions
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Iron Chelators
▪ molecules that bond to iron and may transport it
▪ may be a key component in treatment of iron abnormalities
▪ Types of Chelators
▪ natural and weaker e.g. ascorbic acid may help with minor iron deficiency and be helpful as an adjuvant to oral supplements of iron
▪ Bacteria produced e.g. siderophores like pyoverdine
▪ Phytochemicals with chelating properties e.g. quercetin and rutin
▪ Synthetic iron chelators used in treating thalassemia (iron overload)
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Synthetic Iron Chelators▪ Five main: deferiprone (L1), deferoxamine (DFO), deferasirox (DFRA),
ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetic acid (DTPA)
▪ L1 was used because it is one of the safest and effective treatments for thalassemia and is not produced by bacteria (i.e. DFO)
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Flavonoids
▪ one of the most common phytochemicals found in plants
▪ catechin = green tea
▪ quercetin = buckwheat, green tea, berries, etc.
▪ Metabolic, insecticide, antifungal properties
▪ Found to exhibit strong antioxidant properties towards iron [3]
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Iron Chelation Therapy
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Variables/Control
▪ The variables include the following: the concentration of iron in the media, the type of chelator added to treatments, and the concentration of chelator added to treatments.
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Hypothesis
▪ The flavonoids, quercetin and catechin, and the iron-chelating drug, L1, are predicted to have inhibitory effects on the growth of P. fluorescens in both concentrations of iron media. Siderophoreproduction is expected to positively correlate with bacterial growth
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Materials
▪ 100 mg Deferiprone (L1) (UCLA)
▪ 1 g Catechin Hydrate ( 96 %, Sigma Aldrich)
▪ 1 g Quercetin (98%, Samsara Herbs)
▪ Slant culture P. fluorescens (FlinnScientific, Inc)
▪ 1 g iron (iii) citrate (Flinn Scientific, Inc)
▪ 50 ml DMSO (Flinn Scientific, Inc, Catalog)
▪ M9 minimal media (Na2HPO4, NH4Cl, KH2PO4, NaCl, MgSO4, CaCl2, 10% glucose)
▪ 2 and 3 mL microcentrifuge tubes
▪ Analytical balance
▪ Electronic Plate Reader (accuSkanFC)
▪ Spectrophotometer (V5000 Visible
▪ Spectrophotometer)
▪ Centrifuge (Eppendorf Centrifuge 5418
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Methods
M9 Minimal Media Preparation
▪ M9 minimal media was prepared (Na2HPO4, NH4Cl, KH2PO4, NaCl, MgSo4, CaCl2, 10% glucose)
▪ P. fluorescens running culture and a standard curve was made
▪ 400 µM and 4 µM iron media preparation
▪ a stock solution of 0.1 M iron (iii) citrate was serially diluted in the M9 minimal media to make 400 µM and 4 µM iron media
Chelator Preparation
▪ 1 mL of each desired flavonoid concentration (0.2 mM, 0.5mM, 1 mM, 1.5 mM) was dissolved in 100% DMSO at 10 x concentration and was added to treatments at 1 x concentration
▪ 1 mL of each L1 concentration (0.2 mM, 0.5 mM. 1 mM, 1.5 mM) was dissolved in warm water at 10x concentration, and added to treatments at 1 x concentration
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Methods
Optical Density Analysis
▪ the OD of each sample was taken using a spectrophotometer and/or a plate reader at ≈600 nm after 24 hours and compared to the growth curve in order to determine growth inhibition
▪ the samples were centrifuged and the OD of the supernatant was taken at 405 nm using the plate reader to determine pyoverdine presence
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Title and content layout with chart
0
1
2
3
4
5
6
Category 1 Category 2 Category 3 Category 4
Series 1 Series 2 Series 3
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Two content layout with table
▪ First bullet point here
▪ Second bullet point here
▪ Third bullet point here
Group 1 Group 2
Class 1 82 95
Class 2 76 88
Class 3 84 90
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Two content layout with SmartArt
▪ First bullet point here
▪ Second bullet point here
▪ Third bullet point here
Group A
Group B
Group C
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