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TRANSCRIPT
“I’m a medical doctor and an illustrator. I visualize science.”
Dr. Guido Hegasy
www.hegasy.deScientific IllustrationMedical Microbiologist
Contents1 Schistosomiasis2 Multiple Sclerosis3 Hand Disinfection4 Skin Layers5 The Cardiac Cycle6 CRISPR-Cas97 Complement8 EHEC in Hamburg9 Immune Response
1 2 3
5 6
7 8
4
9
Life Cycle of Schistosoma mansoni
Releaseof Cercariae
Release ofMiracidia
Hatchingof EggsInfection of Snails
Release of Eggs withFeces and Urine
AdultSchistosoma Egg Movement
to Gut andUrine Bladder
Worm Pairing
EggProduction
Migration ofSchistosomulae
Skin Penetration
Illustrationhegasy.de
Double Vision
Single-SidedVision Loss
Symptoms and Findings in Multiple Sclerosis
Intention Tremor
Internuclear Ophthalmoplegia
Periventricular White Matter Lesions
Weakness, Paresis, Walking
Impairment
SpasmsBabinski SignIncontinence
Blurred Vision
Illustrationhegasy.de
3x
Hand Disinfection
Illustrationhegasy.de
A
BC
D
EF
G
H
Thick Skin (e.g., Palm)
1
2
3
4
5
7
12
1
3
4 7
9
12
6 8
10
11
Thin Skin (e.g., Axilla)
AC
D
EF
G
H
Layers, Tactile Receptors, and Appendages of Human Skin
B – Stratum lucidum
A – Stratum corneum
C – Stratum granulosum
E – Stratum basale
D – Stratum spinosum
F – Stratum papillare
G – Stratum reticulare
H – Subcutis
Cutis Ep
ider
mis
Derm
is
10 Hair Follicle
M. arrector pili11
Adipose Tissue12
Merkel Cell3
Meissner Corpuscle2
Ru�ini Corpuscle4
Free Nerve Ending1
Pacinian Corpuscle5
Root Hair Plexus6
Holocrine Gland9
Eccrine Gland7
Apocrine Gland8
Arteriole
Venule
Nerve
Concept & Illustration:
www.hegasy.de
Illustration: www.hegasy.de
The Cardiac Cycle
Vena cava inferior
Vena cava superior
Truncus pulmonalis
Aorta
Valva bicuspidalis
Valva aortae
Valva trunci pulmonalisChordaetendineae
Venae pulmonales
Arteriae pulmonales
Atrium dextrum
Atrium sinistrum
Musculuspapillaris
Truncus brachiocephalicus
Arteria carotis communis
Arteriasubclavia
Ventriculus dexter
Valvatricuspidalis Ventriculus
sinister
A.p.
V.p.
1
3
24
Mitral & Tricuspid Valve
Pulmonary &Aortic Valve
Ventricular Systole
Ventricular Diastole
Valve plane
Ventricular Ejection closed open2
Isovolumic Contraction closed closed1
Isovolumic Relaxation closed closed3
Ventricular Filling open closed4
ECG
Syst
ole
Dias
tole
Origin and Function of
CRISPR-Cas9 Technology
Introduction to CRISPR Biology:
Cas9 IntroducesTargeted DNA Breaks
Panel– 4 –
Origin and Function of
CRISPR-Cas9 Technology
Panel– 5 –
Origin and Function of
CRISPR-Cas9 Technology
Panel– 6 –
Exploiting Endogenous DNA Repair Mechanisms
Introduction to CRISPR Biology:
CRISPR-Cas9 Technology Allows Gene Engineering
Introduction to CRISPR Biology:
Double-Strand DNA BreaksAllow Gene Editing
NHEJ
Insertion Deletion
HDR
Template Integration
Gene Disruption Gene Modification
Gene Editing
Fields of Applicationfor Cas9 Technology
Plan
ts Animals
H u m a ns
By joining tracrRNA and crRNA derived sequences with a linker loop, a simplified programmable tool for DNA manipulation is created. The tracrRNA-crRNA-chimera is called a single guide RNA (sgRNA). sgRNA can be easily synthesized. The complex, consisting of Cas9 and sgRNA, scans DNA for the presence of a protospacer adjacent motif (PAM). For Cas9 from Streptococcus pyogenes, this is a 5′-NGG-3′ sequence. When a PAM sequence is detected, the complementary DNA strand is compared to the crRNA derived guide region. If these sequences match, the DNA double strand is cleaved ~3 bp away of the PAM. This introduces a double-strand break (DSB).
With both domains located in the NUC lobe of Cas9, the HNH domain cuts the strand complementary to the guide sequence (target strand) while the RuvC domain cuts the opposite strand. A DSB introduced precisely at a desired genetic site provides a tool for targeted genetic manipulations.
References: Nishimasu H et al. CELL 2014, Hsu P et al. CELL 2014
When a double-strand break (DSB) is introduced in a cell’s DNA, there are two major repair pathways. In one pathway the blunt DNA ends are joined in a process called non-homologous end joining (NHEJ). This mechanism is error-prone and may produce insertions or deletions of bases. These alterations are collectively called Indel mutations. Indel mutations may result in a loss of function of the a�ected gene, e.g., by introducing a frameshi� and/ or a premature stop codon.
Another pathway is called homology directed repair (HDR). This pathway uses a DNA template to repair the site where the DSB occurred. When exogenous DNA is added, the sequence may serve as a template and is then integrated into the repair process. This may lead to the introduction of a new genetic sequence. Both repair pathways may result in a genetic alteration. Thus, targeted introduction of DSBs opens the door for gene editing.
Reference: Doudna JA and Charpentier E SCIENCE 2014.
By introducing double-strand breaks into a living cell’s genomic DNA, CRISPR-Cas9 technology provides a potential molecular tool. This technology enables precise targeting of genes in live organisms to render them accessible to modifications. This technique has already been shown to work successfully in a variety of species and seems to be applicable to virtually any organism.
In maize plants, CRISPR-Cas9 technology has been employed to modify genes to increase herbicide resistance. In mice, a multitude of mutants have been produced, o�en serving as models to analyze human diseases. Genetic modifications in animals are feasible in humans too. Data have already been published from experiments on human embryos. For this reason, a group of scientists, led by those who have invented the technique, have called for a public discussion on the responsible use of CRISPR-Cas9 technology.
Reference: Baltimore D et al. SCIENCE 2015.
Cas9 as a Molecular Tool
Dr. med. HegasyLife Science Illustration
Dr. med. HegasyLife Science Illustration
Dr. med. HegasyLife Science Illustration
PAMRuvC
HNH
sgRNA
LinkerLoop
NUC Lobe
REC Lobe
C3
C2a
C4b C3b
C1qC1-Complex
MASP
C4a
CP-/ LP-Convertase
C4b2a3b
AP-Convertase
C3bBb3b
C3(H2O) C3(H2O)B+FD
C3(H2O)Bb+P
FB FDBa
C2
C4
C2a
C4b
C9C9
C9
FD
+H2O
C2bC4a
ClassicalPathway
LectinPathway
AlternativePathway
The Complement System
TerminalPathway
MembraneAttack
Complex
Amplification Loop
MBL, Ficolin
C7C6
C2b
C8γ
β α
C5a
P
Bb
C3aC3
C9, C9, C9, …
C3a
C1rC1s
FD
FB
FDBa
C5
C5b
Target Surface
C3a
C3Bb
C3b C3b
C3
Bb
C3b C3b
C9
C9
C9
C5b
C7 C8C6
Properdin
Antibody
MicrobialSugars
Serine Protease
Thioester
C3aC4aC5a
C3b
C5aC3a
Anaphylatoxins
Chemotaxis
Opsonin
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EHEC Outbreak Incidencesin Germany
EHEC Outbreak Incidences in Hamburg
0
10
20
30
40
50
60
70
20 21 22 23 24 25 26 27 28 29
Num
ber o
f Cas
es
Calendar Week 2011
Day of Outbreak Detection
EHEC: Onset of Symptoms
HUS: Onset of Symptoms
EHEC+HUS: Date of Notification
Epicurve
Date
Incidence Distribution
1125Districts
2219
19
114
2.1 – 4.0 Cases / 100000
0.0
4.1 – 6.0
6.1 – 12.0
0.1 – 2.0
>12.0
1.0 – 2.02.1 – 4.04.1 – 12.0>12.0
0.00.1 – 2.02.1 – 4.04.1 – 6.06.1 – 12.0>12.0
City of Hamburg
Late
ncy
[day
s]
0
10
20
30
40
16 - 22 M
ay
23 - 29 M
ay
30 May - 5
June
6 - 12 June
13 - 19 June
20 - 26 June
27 June - 3 July
Q3+1.5 IQR
Median
Q1-1.5 IQR
Q1
Q3
Outliers
Notification Latency
Fenugreek Sprouts
In 2011 an outbreak of enterohemorrhagic Escherichia coli occurred in Germany. The outbreak was largely caused by contaminated fenugreek sprouts. The northern part of Germany was greatly a�ected by the outbreak, and the City State of Hamburg had the highest incidences. Incidences ranged from zero for 22 districts to >12 for four districts. Notification latency increased when the peak of the outbreak had passed. The outbreak was detected when 98 persons had already fallen ill from the infection whereas public health authorities had been notified in only 4 cases. Reference: Tahden M et al., PLoS ONE 2016
EHEC O104:H4 Outbreak in Hamburg, Germany, 2011
Text & Illustration:www.hegasy.de
Bacterium
A bacterium may enter the body through injured skin or by penetrating the endothelia of the respiratory or gastrointestinal tract. This may trigger an immune response.
A bacterium may enter the body through injured skin or by penetrating the endothelia of the respiratory or gastrointestinal tract. This may trigger an immune response.
When crossing endothelial barriers and entering human tissue an antigen-presenting cell (APC), e.g., a dendritic cell (DC), may detect the invader and endocytose bacterial antigens.
When crossing endothelial barriers and entering human tissue an antigen-presenting cell (APC), e.g., a dendritic cell (DC), may detect the invader and endocytose bacterial antigens.
Ii
MHC II
Lysosomes
Endosome
Peptide
Antigen-LoadedMHC II
Endosomes containing bacterial proteins are fused with lysosomes and antigenic peptides are released. These are then loaded on MHC class II molecules to present them on the cell surface.
Endosomes containing bacterial proteins are fused with lysosomes and antigenic peptides are released. These are then loaded on MHC class II molecules to present them on the cell surface.
Mature DCs present antigenic peptides on MHC II to the T cell receptor of naïve CD4 T cells together with co-stimulatory surface molecules. This activates the T cell to proliferate and di�erentiate.
Mature DCs present antigenic peptides on MHC II to the T cell receptor of naïve CD4 T cells together with co-stimulatory surface molecules. This activates the T cell to proliferate and di�erentiate.
MHC II TCR
CD3
CD28
CD80
T CellDC
T cells of the Th1 subtype activate macrophages to phagocytose and digest bacteria. B cells are stimulated to synthesize IgG for opsonization. Neutrophils are activated to act at the site of infection.
T cells of the Th1 subtype activate macrophages to phagocytose and digest bacteria. B cells are stimulated to synthesize IgG for opsonization. Neutrophils are activated to act at the site of infection.
Th1 Cell
Macrophage
B Cell
Neutrophil
IFNγ TNFα
CD40L
The DC migrates through lymphatic vessels to a nearby lymph node. Here, it presents bacterial antigens to T cells that have reached the same lymph node via blood vessels.
The DC migrates through lymphatic vessels to a nearby lymph node. Here, it presents bacterial antigens to T cells that have reached the same lymph node via blood vessels.
CD4
APC
DC T Cell
Lymph Node
1
2
3
4 5 6
A Simplified View on the Generation of a Th1 Immune Response
www.hegasy.de
Scientific Illustration Medical Microbiologist
Rantzaustrasse 33 22041 Hamburg Germany
+49 +40 - 348 618 67 [email protected]
Dr. Guido Hegasy
www.hegasy.de