Download - Laboratory Experimental Research
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Laboratory experimental
research
Evy Sulistyoningrum
Histology department
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Outlines
Introduction
Animal experimental
Culture experimental
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Introduction
Laboratory experimental research : biomedical
research
In vivo : animal study
In vitro culture study
Confounding variables were strictly controlled
Research subject:
Certain Animals
Certain cell/tissue/organism culture
Pre-clinical trial
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Steps in biomedical research
1. Choose the appropriate subject
2. Choose research design models
3. Sample size
4. Animal/sample welfare guarantee
5. Treatments/intervention6. Data Collection
7. Analysis
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Subjects
Animals
Custom animal
Animal model for human condition Genetically modified animal
Human cells/ cell model for human cells
Pathogens Microbes
Paracytes
Fungus, etc
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Research designs
Post test-only with control group
Pre and post test with control group Post test-only without control
Pre and post test with control group
Complete Randomized Design
Control? No treatment at all
Disease model
Gold standard
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Post test-only with control group
scheme
T : (X) O1
C : (-) O2
T : treatment group
C : Control group
(X) : given treatment
(-) : no treatment or given placebo
O : observation in treated group
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Pre and post test with control group
scheme
T : O1 (X) O3
C : O2 (-) O4
T : treatment group
C : Control group
(X) : given treatment
(-) : no treatment or given placebo
O : observation
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Simple Complete randomized design
N animals
C
(-)
O
A
Xa
O
B
Xb
O
C
Xc
O
C : Control group (-) : no treatment or given placebo
(X) : given treatment O : observation
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Pre and post test CRD
N animals
C
O1
(-)
O2
A
O1
Xa
O2
B
O1
Xb
O2
C
O1
Xc
O2
C : Control group (-) : no treatment or given placebo
(X) : given treatment O 1 : observation before treatmentO2 : observation after treatment
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Sample size
Animal
Federer : (n-1)(t-1) > 15
WHO Minimal numberReduction
Cells
Literature based
Pathogens
Literature based
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Treatments
Variant depend on research intention
Routebased on research intention
Dosing :
Toxicology : LD50 , etc
Other dosing: literature-based
Human doseneed conversion
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Data collection
Based on research design
Based on data specification
Source: Animal behaviors
Parameters of animal specimen
Animal tissue
Specific products
Can be measuredresearch variables
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Types of Observations
Activity Level e.g., hypoactivity, hyperactivity, restlessness, lack of
inquisitiveness
Attitude e.g., arousal, depression, awareness of surroundings
Behavior, Spontaneous e.g., vocalization, self-trauma, isolation from cage mateswithout disturbing the animal
Behavior, Provoked e.g., vocalization, hiding, aggressiveness, minimal responsewith disturbing the animal
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Types of Observations Body Condition
e.g., missing anatomy
Food and Fluid Intake
e.g., elimination of feces and urine
Skin
e.g., cyanotic, pale, or congested mucousmembranes or skin (ears, feet, tail); skin lesions
Eyes
e.g., clarity/condition of lens, cornea; position ofglobe; condition of eyelids, encrustation
Posture
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Types of Observations
Locomotion e.g., gait, ataxia, action of each limb, position of tail when ambulating
Neurological e.g., tremor, convulsion, circling, paralysis, head tilt, coma
Vital Signs e.g., respiratory distress (open mouth breathing, pronounced chest
movement)
Other clinical parameters that are relevant e.g., presence and status of tumors, infection, or surgical wounds
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Examination
Behavior
Body weight
Surface lesions (wounds, masses)
Hydration status
Body temperature (telemetric methods)
Blood parameters
Other specimens parameters
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Variables & Analysis
Variables
Based on research designs and observation needed
Scalesbased on research preference
Specimen source must be stated
International unit or other common measurement units
Analysis
Based on research objective
Based on variables scales
Descriptive
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Examples
Nodul size
Blood level of AST, ALT, ALPreflect liverfunction
Blood level of ureum, creatininreflect renalfunction
Fasting glucose level, HbA1c
Inhibition zonereflect antibacterial activity
Balooning degeneration
Johnsons criteria for spermatogenic level
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Animal experiments
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Why perform animal experiments?
Some experiments cannot be performed on humansor better performed on animals
Organs and body systems similar to humans andother animals
Susceptible to the same diseases that affect humans
Short life spanallows to be studied throughoutentire life
Environment easily controllable to keep
experimental variables to a minimum
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Benefits of Animal Research
Penicillin Mice
Blood Transfusions
Dogs
Tuberculosis Medicine Guinea pigs
Meningitis Vaccine Mice
Kidney Transplants Dogs and Pigs
Breast Cancer Treatments Mice, Rats and Dogs
Asthma Inhalers Guinea Pigs and Rabbits
Polio Vaccine Mice
Insulin for Diabetics Dogs
Deep Brain Stimulation forParkinson's Disease
Monkeys
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Benefits Continued Vaccine for Smallpox
Vaccine for Anthrax
Rabies Vaccine
Typhoid Vaccine Cholera Vaccine
Treatment for Beriberi
Treatment for Rickets Corneal Transplants Local Anaesthetics
Discovery of Vitamin C Canine Distemper Vaccine Coronary Bypass Operation German Measles Vaccine
MMR Vaccine Antidepressants and Antipsychotic CT Scanning for Improved Diagnosis Chemotherapy for Leukaemia
Medicines to Treat Ulcers Inhaled Asthma Medication Combined Therapy for HIV infection
Medicines for Type 2 Diabetes Cervical Caner Antibodies Avian Flu Vaccine Malaria Vaccine
Modern Anaesthetics
Tetanus Vaccine
Diphtheria Vaccine
Anticoagulants Streptomycin
Kidney Dialysis
Whooping cough Vaccine Heart Lung Machine Hip replacements
Cardiac Pacemakers High Blood Pressure Medicines Replacements of Heart Valves Chlorpromazine Psychiatric Medicine
MRI Scanning for improved Diagnosis Prenatal Corticosteroids for Premature Babies Treatment for River Blindness Life Support for premature Babies
Medicines to control Transplant Rejection Hepatitis B Vaccine Leprosy Treatment
Oral and Inhaled Insulin for Type 1 Diabetes Angiogenesis Inhibitors for Cancer and Blindness Gene Therapy for Muscular Dystrophy Alzheimers Vaccine
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Limitations of animal experimentation
Species differences
Some symptoms are hard to discover in animals
In some cases, genetically modified animals isbetter
discovery of gene functions
treatment and knowledge of genetic disease
minimisation of rejection followingxenotransplantation
development and production of therapeuticprotections
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Hazards of Working with animal
Injuries
Allergies Zoonoses
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Two Major Points of View
(1) Animal Rights- ending all animal use
no food, clothing, entertainment, medical research or hunting
(2) Animal Welfare - animals must be treated and
used humanely
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Rules for animal experiments
Animal Welfare Act, 1966 [USC Title 7, Sections 2131 to 2156] as amended in1970, 1976, 1985 and 1990.
Animal Welfare Regulations [Title 9 CFR, Subchapter A, Animal Welfare, Parts 1, 2and 3]
Health Research Extension Act, 1985 [Public Law 99-158, November 20, 1985,Section 495]
US Government Principles for the Utilization and Care of Vertebrate Animals Usedin Testing, Research, and Training, 1985
PHS Policy on Humane Care and Use of Laboratory Animals, 1986
2000 Report of the AVMA Panel on Euthanasia [JAVMA, Vol. 218, No. 5, March 1,2001]
Guide for the Care and Use of Laboratory Animals (Guide) [NRC, 5th Ed., 1996]
NIH Grants Policy Statement (03/01), Part II: Terms and Conditions of NIH GrantAwards Subpart A: General -- Part 2 of 7
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The 3 Rs
Russel & Burch (1959):
(a)Refinement: Improve the experiments forminimising animal pain and suffering
(b)Reduction: Use statistical methods so that a
smaller number of animals are required(c)Replacement: Use alternative, non-animal methods to achieve
the same scientific aim
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Refinements
Proper animal living conditions
Qualified and trained personnel
Medical care available and provided by a qualified
veterinarian Procedures avoid or minimize discomfort, distress, andpain
Procedures that may cause pain or distress : Analgesia unless well justified Unrelieved paineuthanize
Surgical procedures performed aseptically andappropriate
Animals not used in more than one major, survivaloperative procedure
Acceptable methods of euthanasia
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Examples of Refinement
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Reduction
Animal Numbers : minimum possible to achieve
statistically significant data and are well justified :
Citation of previous research: sufficient information toindicate that the previous research is similar enough in
concept and methodology
Power analysis: enough information to show ability in
analyzing data and using a power analysis
If need animal specimen: state clearly how many
amount of material is needed
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Replacement
In Vitro Testing
Computer Modelling
MRI Scanning
Micro dosing
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Animal models
Animal that biologically or habitually normative, canbe spontaneously or patogenetically investigated,and human phenomenon mimicking
Mice: most frequently used (mice + rodents :+ 90 %)
Other rodents: Rats
Guinea pig
Hamster etc
Dogs, cats, rabbits, farm animals, fish, frogs, birds,nonhuman primates, etc : 10%
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Animal models
Human condition mimicking: Diabetes
Dislipidemic
Hepatotoxic Ulcers
Cancers
Dosing Literature based
Conversion from human dose
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Animal handling
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Acclimation & sexingAcclimation
Period of time allows animals to adapt to a newenvironmentbiological stabilization
Effects of transportation stress : various blood parameters
food intake and animal behavior
Period of time necessary : depend on theparameters to be studied.
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Sexing
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Sexing
scrotum
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Mouse Pups
1 day old mice 2 days old pups day 3
4 days old pups 6 days old pups5 days old pups
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and more pups
Day 7 Day 8 Day 9
Days 12,13 and 14Day 11Day 10
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Blood Collection from Tail
TOOLS
Alcohol cotton ball forsurface disinfection
Small plastic bottlewith 1/2 cm diameterholes in both ends asmouse restrainer
Scissors Micropipette and tips
A vial for bloodcollection
For collection of small amount ofblood (Approximate 0.1 ml )
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Leave the tail of the mouseoutside the cover of the
restrainer
Amputate the tip of the
mouse tail by scissors
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Massage the tail and collect blood by pipette
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Blood Collection from Orbital Sinus in
Mouse
TOOLS Alcohol cotton ball for
surface disinfection
Kloroform for generalanesthetic
27 G needle with 1 mlsyringe for injection
Glass capillary tubeand vial for blood
collection
Anesthetic before blood withdraw
Collect amount up to 0.5 ml
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Anesthetize a mouse byintraperitoneal injection
Use a glass capillary tube to
penetrate the orbital conjunctiva
and rupture the orbital sinus
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Collect blood with a vial
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Cardiac puncture in Mouse
TOOLS
Alcohol cotton ball forsurface disinfection
Chloroform used asanesthetic
27G needle with 1 mlsyringe for injection
24G needle with 3 mlsyringe for bloodwithdraw
For collect up to 1 ml of blood within a shortperiod of time
Must be performed under general anesthetic
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Anesthetize a mouse by
intraperitoneal injection
Disinfect the thorax area
with 75% alcohol cotton
ball
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Search for the maximum
heart palpitation
Cardiac puncture in 90
angle
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Withdraw blood slowly by right hand
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Blood collection from Saphenous vein in mice
TOOLS Alcohol cotton ball
for surfacedisinfection
50 ml syringe tubewith small holes atthe end asrestrainer
a scalpel and shaverfor remove of hair
24 G 1 needle forrelease of blood
tips and pipette forblood collection
Multiple samples taken in the course of aday
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Place the mouse in the
restainer
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Pull out the leg and
removed the hair by a
assistant
Hair also be shaved by
using a small scalpel
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The saphenous veinis seen on the surface
of the thigh
Apply vaseline to the
surface area to reduceclotting and coagulation
during blood collection
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The saphenous veinis ruptured by scalpel
Blood is aspirated
through pipette
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Injection in Mouse
TOOLS
Alcoholcotton ballfor surfacedisinfection
25G 1/2needle with1 ml syringe
for injection
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Handle a mouseproperly
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The injection site : the lower left quadrant of the abdomen
because vital organs are absent from this area. Only the tip of
the needle should penetrate the abdominal wall to prevent
injection into the intestine.
Intraperitoneal injection
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Pick up a nude mouse and spin its tail
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Subcutaneous injection
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Oral Feeding in Mouse
TOOLS
A 18 G
stainlesssteel, balltipped
needle a glove
Gastric intubation ensures that all the material wasadministered
Feeding amount limited to 1% of body weight
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Gastric intubation
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Dissection
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Genetically modified animal
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Transgenic mice
Transgenic: an organism that has had DNA
introduced into one or more of its cells artificially
transgenic: DNA is integrated in a randomfashion by injecting it into the pronucleus of a
fertilized ovum
Random (+ 10% disrupt an endogenous gene important
for normal development)
multiple copies
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Knock-out mice
knockout: DNA is introduced first into embryonic
stem (ES) cells
ES cells that have undergone homologous
recombination are identified and injected into a 4 dayold mouse embryo - a blastocyst
targeted insertion
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Pronuclear injection in transgenic
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Pronuclear injection in transgenic
technology
Implanting 1(or 2) cell embryos
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Implanting 1(or 2) cell embryos
(cont )
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(cont.)
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Transgenic mice
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Trangenic mouse embryo in which the promoter for a gene
expressed in neuronal progenitors (neurogenin 1)
drives expression of a beta-galactosidase reporter gene.
Neural structures expressing the reporter transgene are dark
blue-green. (Dr. Anne Calof)
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Tail tip9.5 day embryos -
GFP and wt
GFP transgenic mouse (Nagy)
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GFP transgenic mouse (Nagy)
ES cells growing in culture in knock out
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ES cells growing in culture in knock out
technology
Successfully transformed ES cells are
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Successfully transformed ES cells are
injected into blastocysts
l bl
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Implanting blastocysts
1 2
l bl ( )
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Implanting blastocysts (cont.)
3 4
i
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Littermates
Black mouse -
no apparent ES cell
contribution
Chimeric founder -
strong ES cell
contribution
Chimeric founder -
weaker ES cell
contribution
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Cell culture
I d i
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Introduction
Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under controlled
conditions In practice it refers to the culturing of cells derived
from animal cells.
Cell culture was first successfully undertaken by Ross
Harrison in 1907
Genetic engineering can be done to cellsproduce
desired substance (recombinant)
E l f bi t
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Examples of recombinant
products
Viral vaccine
Hybridoma capable of secreting a monoclonal
antibody.
Human insulin recombinant protein
Human growth hormone produced fromrecombinant bacteria
Lymphoblastoid IFN
Tissue-type plasminogen activator (tPA) from
Wh i ll lt d f ?
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Why is cell culture used for?
Model systems for:
Studying basic cell biology
Interactions between agents and cells
Effects of drugs on cells
Toxicity testing
Study the effects of new drugs on cells
Cancer research
Study the function of various chemicals, virus & radiation
to convert normal cultured cells to cancerous cells
Study of the effects of drugs in cancer cells
Contd
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Contd.
Virology
Cultivation of virus for vaccine production
Genetic Engineering
Production of commercial proteins
Gene therapy
Cells having a functional gene can be replacedto cells which are having non-functional gene
P i lt
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Primary culture
Cells when surgically or enzymatically removed
from an organism and placed in suitable culture
environment will attach and grow
Primary culture contains heterogeneous
population of cellssub culturing of primary
cells leads to generation of cell lines
Cell lines have limited life span, they passage
several times before they become senescent
S C ll li
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Some Common cell lines
Human cell lines
MCF-7 breast cancer
HL 60 Leukemia
HEK-293 Human embryonic kidney
HeLa Henrietta lackscervical cells infected by HPV 16
HUVEC human Umbilical Vein Endothelial cells
Primate cell lines Vero African green monkey kidney epithelial cells
Cos-7 African green monkey kidney cells
C t i t f ll lt
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Contaminants of cell culture
Chemical:
difficult to detectinvisible
caused by endotoxins, plasticizers, metal ions or traces of
disinfectants
Biological:
cause visible effects Mycoplasma, yeast, bacteria or fungus or cross-
contamination of cells from other cell lines
Basic equipments used in cell culture
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q p
Laminar cabinet
Incubation
Refrigerators
Inverted microscope
Tissue culture ware- Culture plastic waretreated by polystyrene
P th lt
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Pathogen cultures
Similar with cell culture
Larger organism
Proper & specific media
McConkey
Sabouroud
etc
Can be treated with various substance
produce specific effect that can be
measurable
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