![Page 1: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/1.jpg)
Pathways of Skeletal Muscle Atrophy: HIV as
a Model System?
Chelsea Bueter, Michelle McKinzey, Chloe Salzmann, Michael Zorniak
Department of Biology, Lake Forest College, Lake Forest, Illinois 60045 USA
![Page 2: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/2.jpg)
Presentation Pathway
Introduction
Diseases
HIV
Cachexia
Oxidative Stress
Diabetes
Discussion
![Page 3: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/3.jpg)
Paradigm of SMAHypertrophy vs. Atrophy
http://www.nndb.com
Introduction
![Page 4: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/4.jpg)
Most Studied Pathway
Stress
PI3K
AKT
FOXO
mTOR
Atrogin-1
Protein degradation
Protein synthesis
S6K
(Nucleus)
pp
Introduction
![Page 5: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/5.jpg)
HIV
Skeletal Muscle Atrophy
Diabetes
VPR Protein
Oxidative Stress
Cachexia
??
?
? ?
?
?
?
![Page 6: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/6.jpg)
The Past of HIV
• Vpr protein stops the cell cycle
• Prevents programmed cell death
• Increased replication of HIV
• AIDS muscle wasting symptom
Introduction
![Page 7: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/7.jpg)
HIV to SMA?
• Vpr secreted like a hormone
• Infects other cells and organs
• AIDS wasting syndrome in skeletal muscle
Introduction
![Page 8: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/8.jpg)
Cancer Cachexia
http://www2.msstate.edu/~shbryd/Disorders.html
• Cachexia is a syndrome in cancer patients
• Progressive muscle wasting, weight loss, weakness and fatigue
Introduction
![Page 9: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/9.jpg)
The Past of Cachexia
• Pathway unknown
• Cytokines induced muscle wasting
• One hypothesis: Muscle wasting in cancer due to increased energy consumption
Introduction
![Page 10: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/10.jpg)
What is Oxidative Stress?
• Cancer, Parkinson’s, Diabetes, and SMA• Free Radicals or Reactive Oxygen Species• Steal electrons to restore valence stability
Introduction
![Page 11: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/11.jpg)
The Past of Oxidative Stress
Goldberg et al, 1986
• Calcium activates protein degradation
Appel et al, 1997
• Vitamin E decreases calcium levels
• Vitamin E is an anti-oxidant
• Thus, oxidative stress calcium levels and activates protein degradation.
Introduction
![Page 12: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/12.jpg)
Diabetes
• Characterized by insulin deficiency or insulin intolerance
• Juvenile diabetes (type 1)- genetically linked but also diet linked
• Type 2 - middle-aged people-low insulin levels
• Leads to many other disorders
Introduction
![Page 13: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/13.jpg)
The Past of Diabetes
• 1993- studies showed that in non-diabetics, insulin levels and activity remained high
• Diabetics showed very low insulin levels or activity
• Common symptom in diabetics was SMA
• Hypothesis= Insulin tolerance may be linked to SMA
Introduction
![Page 14: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/14.jpg)
Presentation Pathway
IntroductionDiseases
HIVCachexiaOxidative StressDiabetes
Therapies Discussion
![Page 15: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/15.jpg)
HIV
?
Skeletal Muscle Atrophy
![Page 16: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/16.jpg)
HIV’s Vpr protein
• Two Direct Pathways to SMA
1. Insulin Resistance
2. Glucocorticoid Hypersensitivity
HIV
![Page 17: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/17.jpg)
Effects of Vpr binding
Vpr
Cdc25
Serine/Threonine residues
Cdc25
14-3-3
14-3-3
Vpr
14-3-3
Vpr
Cdc25
HIV
![Page 18: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/18.jpg)
Vpr Inhibits Cell Cycle
Triggers mitosis machinery
Alberts et al 2004
HIV
![Page 19: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/19.jpg)
Stopped Cell Cycle
G2/MDividing G2/MDividing
HIV
•Vpr stops the cell cycle at G2/M
He et al 1995
![Page 20: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/20.jpg)
14-3-3
Vpr
Vpr inhibits insulin effects on FOXO
Vpr
Cdc25
14-3-3
Cdc25
Serine/Threonine residues on FOXO
HIV
![Page 21: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/21.jpg)
Effect of Vpr on FOXO
HIV
•Vpr doesn’t bind FOXO
Kino et al 2005
![Page 22: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/22.jpg)
What is insulin supposed to do?
FOXO
(Nucleus)
p
Insulin
FOXO p14-3-3
FOXO
Insulin
14-3-3
FOXO p14-3-3
FOXO p
14-3-3
HIV
![Page 23: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/23.jpg)
Vpr won’t let insulin work!
FOXO
No FOXO
HIV
Kino et al 2005
![Page 24: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/24.jpg)
How does Vpr affect glucocorticoid receptors?
Vpr
LQQLL
HIV
•Specific LXXLL motif binds GRE
•Completely different from ability to arrest cell cyle
Kino et al 2000
![Page 25: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/25.jpg)
Vpr as a Co-regulator
Kino et al 2000
![Page 26: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/26.jpg)
Vpr as a Co-regulator of GR
Vpr
TATA
Transcription: enhancing glucocorticoid signal
G R
TFIIB
TFIID
RNA polymerase II
GRE
HIV
Kino et al 2000
![Page 27: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/27.jpg)
Summary of Vpr & SMAVPR Protein
Skeletal Muscle Atrophy
VprGRE
Glucocorticoid
14-3-3
Vpr
FOXOnucleus
Atrogin-1
![Page 28: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/28.jpg)
Therapies for HIV muscle wasting
• Steroid hormone receptor antagonists (RU 486)
• Vpr antagonists
• Current antiretroviral therapies
![Page 29: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/29.jpg)
Cancer Cachexia
Skeletal Muscle Atrophy
?
![Page 30: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/30.jpg)
NF-κB
Nucleus
IκB
NF- κB
IκBα
P
Cachexia
![Page 31: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/31.jpg)
Cai et al. Study
MISR Mouse MIKK Mouse
Constitutively active IκBα
Constitutively active IκB
Cachexia
Inactive NF-κB Always active NF-κB
![Page 32: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/32.jpg)
NF-κB Activity
• NF-κB activity is high in MIKK mice
Cachexia
![Page 33: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/33.jpg)
MIKK Mice vs. MISR Mice
• MIKK mice have a much lower body mass
Cachexia
![Page 34: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/34.jpg)
Tumor Activity
• Presence of a tumor increases the level of NF-κB activity in wild type mice
Cachexia
![Page 35: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/35.jpg)
Tumor Necrosis Factor - TNFα
• In the presence of IκBα, activity decreases
• Without IκBα, inhibitor does not stop production
Cachexia
![Page 36: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/36.jpg)
What does NF-κB affect?
• MURF1 mRNA is much higher in MIKK mice than in MISR mice
Cachexia
![Page 37: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/37.jpg)
What else does NF-κB affect?
• TNF activates NF-κB which causes a decrease in MyoD production
Cachexia
![Page 38: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/38.jpg)
Troponin in Cardiac Muscle
• Troponin-1 is degraded in the presence of MURF1
Cachexia
![Page 39: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/39.jpg)
Cachexia Pathway
TNF-α
NF-κB
MURF1 MyoD
activates
decreasesincreases
![Page 40: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/40.jpg)
Therapy for Cachexia
• Salicylate inhibits the NF-κB pathway, preventing muscle loss
Cachexia
![Page 41: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/41.jpg)
Oxidative Stress
Skeletal Muscle Atrophy
?
![Page 42: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/42.jpg)
Oxidative StressReactive Oxygen Species
Mitochondria
Caspase-3Calpain
Sarcomere
Unit of Myofibril
20S/26S Proteasome
Calcium
Cytochrome c
CalpastatinCalpastatin
NO
Oxidative Stress
![Page 43: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/43.jpg)
Effect of Disuse
• Disuse increases oxidative stress
Tidball et al, 2002
Oxidative Stress
![Page 44: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/44.jpg)
Effect of Oxidative Stress
• Increase of oxidative stress increases calsequesterin
• Calsequestrin sequesters intracellular calciumHunter et al, 2001
Laser Densitometry
Oxidative Stress
![Page 45: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/45.jpg)
Effect of Increased Calcium
Type II Diaphragm Muscle Fibers by Immunohistochemistry
• Disuse increases calpain and 20S proteasome activity
Tidball et al, 2002
Oxidative Stress
![Page 46: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/46.jpg)
Calpain Proteolysis
Koh et al, 2000
• Calcium treatment increases protein cleavage
• Protein cleavage can be inhibited by nitric oxide and calpastatin
Oxidative Stress
![Page 47: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/47.jpg)
Caspase Activity
• Caspases inhibit calpastatin
• Calpastatin is a calpain inhibitor
Wang et al, 1998
Oxidative Stress
![Page 48: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/48.jpg)
SummaryOxidative Stress
Increase Ca2+
Calpain & Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
![Page 49: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/49.jpg)
Therapies for Oxidative Stress
• NO and Calpastatin Transgene• Vitamin E protects against ROS• Vitamin C restores Vitamin E activity
Cell
Vitamin E
ROS
Oxidative Stress
![Page 50: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/50.jpg)
Diabetes
?
Skeletal Muscle Atrophy
![Page 51: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/51.jpg)
Diabetes
Insulin levels
Ub-ligase E3-α
26 S proteasome
![Page 52: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/52.jpg)
What activates the Ubiquitin-proteasome pathway?
• No difference in diabetics without vs. diabetics with acidosis
• Acidosis is not a stimulus of ubiquitin dependent atrophy
Diabetes
Price et al. 1999
![Page 53: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/53.jpg)
Effects of Insulin on Ub-proteasome pathway
• Less protein degradation in insulin treated muscles
• Lower insulin levels leads to activation of Ub-proteasome pathway
Diabetes
Price et al., 1999
![Page 54: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/54.jpg)
Results of Pathway Activation
• Levels of Ub-ligase and its coenzyme increase
• Amount of Ub-conjugation by these increases
Diabetes
Goldberg et al., 1999
![Page 55: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/55.jpg)
Proteasome Formation
• mRNA for 19 S and 20 S subunits increases• Formation of 26 S proteasome increases
Diabetes
Attaix et al., 2004
![Page 56: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/56.jpg)
Proteasome Activity
• Flourescence in atrophied muscles higher than control muscles
• Flourescence is analogous to amount of 26 S proteasome activity
Diabetes
Attaix et al., 2004
![Page 57: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/57.jpg)
Summary of Diabetes and SMADiabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
![Page 58: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/58.jpg)
Therapy for Diabetes
• Treatments for diabetes generally focus on maintaining available insulin levels NOT SMA
• Side effect of the insulin treatment, however, is associated with the reverse pathway of atrophy, i.e. hypertrophy
Diabetes
![Page 59: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/59.jpg)
Presentation Pathway
Introduction
Diseases
HIV
Cachexia
Oxidative Stress
Diabetes
Discussion
![Page 60: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/60.jpg)
HIV
Skeletal Muscle Atrophy
Diabetes
VPR Protein
Oxidative Stress
Cachexia
?
??
![Page 61: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/61.jpg)
Vpr
VPR Protein
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Co-activates glucocorticoid receptor
Atrogin-1 induction
Skeletal Muscle Atrophy
![Page 62: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/62.jpg)
Cachexia
Cachexia
Murf-1 increase
IKK/NF-kappa B pathway
MyoD mRNA decrease
Skeletal Muscle Atrophy
![Page 63: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/63.jpg)
Oxidative StressOxidative Stress
Increase Ca2+
Calpain & Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
![Page 64: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/64.jpg)
DiabetesDiabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
![Page 65: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/65.jpg)
HIV
Skeletal Muscle Atrophy
VPR Protein
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Co-activates glucocorticoid receptor
Atrogin-1 induction
Oxidative Stress
Increase Ca2+
Calpain & Caspase 3
activity increases
Releases Actomyosin
to be degraded in proteasome
Cachexia
Murf-1 increase
IKK/NF-kappa B pathway
MyoD mRNA decrease
Diabetes
Insulin decrease/ glucocorticoid
increase
E3-alpha ubiquitin increases
26 S Proteasome activity increases
![Page 66: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?](https://reader035.vdocument.in/reader035/viewer/2022062309/568147ab550346895db4e60f/html5/thumbnails/66.jpg)
Acknowledgements
Thanks to Dr. D, Sara Herrera, Tammy
Hibler, Arun Paul, and Chris Prater