“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

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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

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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

Illustrationhegasy.de

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

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Scientific Illustration Medical Microbiologist

Rantzaustrasse 33 22041 Hamburg Germany

+49 +40 - 348 618 67 info@hegasy.de

Dr. Guido Hegasy

www.hegasy.de