impacts(offood(on(( …...• cell/tissue bioassays & electrophysiology • animal models core...
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IMPACTS OF FOOD ON INTESTINAL FUNCTION & HEALTH
JULIE DALZIEL, SENIOR RESEARCH SCIENTIST, FOOD NUTRITION GENOMICS AGRI-‐FOODS & HEALTH
TOKYO, 12 OCTOBER 2010
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AGRESEARCH
NZ’s largest Crown Research Institute • Wholly government-owned • 860 full-time equivalent staff (250 PhDs)
Research and development funding • 45% revenue from competitive grants • 55% revenue from commercial contracts • Total annual revenue (08/09): NZ$ 148m • Small amount of direct Government funding
Research and development scope • From “paddock to the plate” • Basic and applied research
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OUR FOCUS
Raise productivity in the NZ pastoral sector in an environmentally sustainable manner.
Introduce a range of biotechnologies and other technologies to NZ.
Export our own biotechnologies and other technologies to the world.
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AGRESEARCH SITES:
Ruakura (Hamilton)
Grasslands (Palmerston North)
Wallaceville (Wellington)
Lincoln (Christchurch)
Invermay (Dunedin)
+ several farms around NZ
AgResearch Grasslands Palmerston North
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AGRESEARCH SCIENCE STRUCTURE
CEO Tom Richardson
GM – Food & Textiles Warren McNabb
Animal Health Bio-based Products & Textiles
Rumen Nutrition & Microbiology
GM – Applied Biotechnologies Jimmy Suttie
Forage Biotechnology
Animal Improvement
Animal Biosciences
Bioinformatics, Mathematics & Statistics
Forage Improvement
GM – Agriculture & Environment Peter Benfell
Biocontrol & Biosecurity
Agricultural Systems
Climate, Land & Environment
Agri-Foods & Health
Greenhouse Gas Research Centre
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NutriLon Chemistry
Microbiology
Metabolism
AgResearch’s Systems Biology Platform
Proteomics Metabolomics
PlaNorm
FuncLonal (Epi)genomics PlaNorm
BioinformaLcs MathemaLcs Modelling PlaNorm
Meat Science Food Safety
Food Nutrition Genomics Dairy Foods Food Microbiology & Safety Meat Science & Technology
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DAIRY FOODS -‐ PROTEIN PURIFICATION
Purification of proteins and milk fractions • lab-scale (mg to gram quantities) • pilot-scale (gram to kg)
Anti-fungal, anti-viral, anti-bacterial e.g. nutraceutical food, crop protection, food preservative
(Smolenski et al., 2007, J Proteome Res 6: 207-15)
Immune-modulating e.g. nutraceutical food
Enzymes for flavour development e.g. kokumi
Mold growing on food and crop products
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Team Leader • Dr Nicole Roy
Team composition • 12 scientists • 8 technicians • 11 PhD students
Core capabilities • Functional genomics • Microbiology • Cell/tissue bioassays & electrophysiology • Animal models
Core areas of research • Food, host and microbial interactions • Molecular Nutrition (nutritional genomics, epigenomics, metabolomics) • Neuromotor function and membrane physiology
FOOD NUTRITION GENOMICS TEAM
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The intestinal tract is a highly complex system – food/microbe/mucosal interface
Human intestinal mucosa • Largest interface (barrier) between
humans and the environment. • Critical for the balance between
health and disease. • Crucial to food utilisation; poor
function linked to diet-related diseases.
>25,000 food components
Foods can affect: • probiotic bacterial growth • immune system balance • intestinal barrier function • intestinal motility
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What is intestinal barrier function? Intestinal barrier separates intestinal lumen from underlying tissue and provides a protective interface between internal and external environment of body.
Physical barrier (the epithelium)
Chemical barrier (mucus layer)
Immunological barrier (immune cells of the lamina propria)
Microbial barrier (commensal bacteria)
Hooper LV (2009) Nat Rev Microbiol. 7(5):367-74.
Muscle layers (smooth muscle gut wall)
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Intestinal muscle assay
Fluorescence-based cellular assay
Intestinal permeability assay
Patch-clamping
CELL & TISSUE BIOASSAYS Techniques to measure changes in cell and tissue function in response to food/pharmaceuticals and therefore predict human health outcomes and detect unwanted gastrointestinal side effects.
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INTESTINAL SMOOTH MUSCLE Intestinal contraction is determined by circular and longitudinal muscle layers and affected by neuronal inputs
ORAL
NEUROMOTOR FUNCTION
Interneuron Longitudinal
muscle motor neuron
Circular muscle
motor neuron
Interneuron Network of neurons
ANAL
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Measures effects of intestinal contents on smooth muscle contractility
Provides information on whether intestinal contractility is affected.
INTESTINAL MUSCLE ASSAY
Con
tract
ion
forc
e
Control
Test condition
Control
Time
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Indirectly measure changes in membrane permeability by detecting changes in membrane potential or intracellular calcium.
Provides general information on whether receptor/ion channel mechanisms are affected
Permeability measured across the cell membrane (fluoresense, patch-clamp), between cells, or both (TEER).
FLUORESCENCE-BASED CELLULAR ASSAY
apical
basolateral
Cl K
Na2Cl
K
K
2K
ATP
3Na
Cl K
Na2Cl
K
K
2K
ATP
3Na
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TEER measures changes in tight junction resistance across an epithelial Caco-2 cell monolayer using an ohmmeter
Example shows a control, a bacterial strain that decreases epithelial resistance (strain 2), and another that increases resistance (strain 1).
The results indicate improved (increased resistance) or weakened (decreased resistance) epithelial barrier function by bacteria (food ingredients could also be used).
TRANS-EPITHELIAL ELECTRICAL RESISTANCE ASSAY (TEER)
Control Strain 1 Strain 2
bacteria added
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ION CHANNELS IN THE CELL MEMBRANE
cellular excitability
muscle contraction, neuronal firing and neurotransmitter release
intestinal motility
cellular permeability
ion and water absorption/secretion
faecal moisture
faecal consistency (diarrhoea/constipation) - microbial balance - enteric nervous system - immune system
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Patch-clamping of cells to directly measure ionic currents across cell membranes as ion channels open and close.
ELECTROPHYSIOLOGY
Express protein Identify cell Record current
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Patch-clamp data example of an inhibitory effect measured for a K+ ion channel
ELECTROPHYSIOLOGY
Dose response relationship
control test condition recovery
Provides specific information on how receptor/ion channel mechanisms are affected.
Dalziel et al. 2005, Toxicology Letters 155: 421-6.
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RELEVANT PUBLICATIONS
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COLLABORATORS NZ: Auckland University, Liggins (Auckland), Plant & Food Research, Otago
University (Dunedin), Massey University Riddet Institute (Palmerston North).
International:
A/Prof Andrea Meredith, University of Maryland – K+ channel knockout mice
Prof Shunyi Zhu, Chinese Academy of Sciences – K+ channel inhibitors
Dr Yue-kun Ju, University of Sydney – Cardiac ion channels
Prof Richard Aldrich, University of Texas – Ion channel research
Prof Kikuji Itoh, Tokyo University – Germ-free rodents.
Prof Jeremy Wells, Wageningen University – Intestinal barrier function.
Prof Ian Rowland, Reading University – Molecular nutrition/cancer.
Prof Jonathan Powell, Cambridge University – Nanoparticles.
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COLLABORATIVE OPPORTUNITIES
Protein purification
Test effects of food ingredients on intestinal function, including motility and effects on receptors and ion channels
Explore underlying mechanisms of action
AgResearch Grasslands
Palmerston North
We invite you to visit us in NZ!