BME2106 - Intro to Cellular and

Biomolecular Engineering
WATER is the solvent of choice
Week 2 for biological systems
Dr. Bee Luan KHOO
blkhoo@um.cityu.edu.hk
Room G6617

Department of Biomedical Engineering

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BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong

o Constitutes 70-85% of cell weight, typically Water is a unique solvent whose properties are
o Important as a solvent and a reactant in biochemical reactions extremely important to biochemistry.
o Helps regulate temperature since it is able to absorb large amounts of heat

o Helps regulate intracellular pH

o Used for transport – delivers nutrients andremoves waste from cells

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 In water,
the hydrogen atoms have a partial positive charge, and
the oxygen atoms have a partial negative charge.
Chemical bondings
Water is a dipole because of its geometry and
the difference in electronegativity between hydrogen and oxygen.
1. Covalent bonds (strong)
2. Ionic bonds (strong)
3. Hydrophobic interaction (strong)
4. Hydrogen bonds (weak)
5. van der Waals force (weak)

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BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong

Covalent bonds Ionic bonds = attractions between ions of

opposite charge
Seen in water molecules, involve the mutual sharing of
electron pairs, creating strong linkages. Ionic bonds result  An ion is an atom or molecule with an electrical charge resulting
from gain or loss of electrons.
from electron transfer between atoms, leading to the
When an electron is lost, a positive charge results.
attraction of oppositely charged ions.


 When an electron is gained, a negative charge results.

 Two ions with opposite charges attract each other.
 When the attraction holds the ions together, it is called an ionic bond.
 Salt is a synonym for an ionic compound.
Transfer of electron

Na Cl
Sodium atom Chlorine atom Na Cl
Sodium ion Chloride ion

Sodium chloride (NaCl)

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BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong
 Hydrophobic interactions provide a common
noncovalent force between two molecules Hydrogen bonds
 Most large molecules are held in their three-dimensional
 Hydrophobic interactions occur when oil-like substances avoid
functional shape by weak bonds.
contact with water. Oil and water do not mix!
 When hydrogen is part of a polar covalent bond, H+ has
 It is usually referred to the driving force responsible for the folding of
proteins. a partial positive charge.
 Hydrophobic interaction is the removal of nonpolar amino acids from The charged regions on molecules
solvent (water) and their burial in the core of the protein. are electrically attracted to
oppositely charged regions on
neighboring molecules.
Because the positively charged
region is always a hydrogen atom,
the bond is called a hydrogen
bond.
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BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong

van der Waals force van der Waals force

How gecko walks on the wall
 Also called van de Waals Millions of tiny hairs on the gecko foot adhere to the wall
through Van Der Waals forces, which are basically small
interaction or London forces attractions between molecules that are close together.
 Arises from random variations
in the positions of the
electrons around one nucleus,
 When two atoms comes
within 5 nm of each other,
there will be slight interaction
between them, causing
polarity and slight attraction.

Dipole-Dipole Forces: Specific type of van der

Waals force, involving attractive forces between
polar molecules.
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BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong
 van der Waals force
Impact: From Nature to Technology Kiel University

[2] Genes and Diseases

light-actuated grippers which are activated simply by shining a UV light on a new adhesive material. The researchers are working towards a
device that will emulate the way geckos seamlessly scurry across precarious surfaces in almost any direction.
https://interestingengineering.com/light-actuated-grippers-inspired-geckos

BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong

Biological Heterogeneity Types

1. Spatial
2. Temporal
Why should we be concerned about cellular 3. Physical
diversity? 4. Genomic
5. Proteomic
6. Functional
 The Importance Of Knowing Cellular
Diversity/Heterogeneity

'If you know the enemy and know yourself, you need
not fear the result of a hundred battles' - Art of War

GE1320: Cellular Diversity BME, City University of Hong Kong

What are the heterogeneity

What are the heterogeneity
factors leading to diversity in
factors leading to diversity?
cancer?
Intratumoural heterogeneity
• Spatial heterogeneity, which describes the uneven
distribution of genetically diverse tumour subpopulations
across different disease sites or within a single disease site
or tumour,
• Temporal heterogeneity, a term applied to the dynamic
variations in the genetic diversity of an individual tumour
over time
 What are the heterogeneity What is the impact of such
factors leading to diversity? heterogeneity?
3. Genomic instability fosters genetic diversity by
providing the raw material for the generation of • Tumours with high levels of intratumoural heterogeneity
might predispose patients to inferior clinical outcomes
tumour heterogeneity
• Under therapeutic selective pressure, resistance to
treatment can emerge as a result of the expansion of pre-
existing subclonal populations or from the evolution of
drug-tolerant cells

1. What are the two types of diversity discussed in this paper?

2. What were the techniques used to evaluate intratumor heterogeneity?
3. What is the impact of such heterogeneity?
4. What can be done to handle intratumor heterogeneity?

GE1320: Cellular Diversity

Schmitt et al Ann N Y Acad Sci. 2012; 1267: 110–116.
BME, City University of Hong Kong
 Causes of Heterogeneity Causes of Heterogeneity
Tumors: A Case Study Tumors: A Case Study
1. Genetic Mutations 1. Genetic Mutations
- Variation in the form of acquired -
mutations Acquired = somatic Acquired = somatic
- Stochastic/Unpredictable genomic • Occur at some time during a person's life • Occur at some time during a person's life
instability, e.g. increased replication • Present only in certain cells, not in every • Present only in certain cells, not in every
errors cell in the body. cell in the body.
- Selection by therapeutics e.g. environmental factors such as e.g. environmental factors such as
ultraviolet radiation from the sun, ultraviolet radiation from the sun,

http://www.md-health.com/images/10422517/image-1.jpg
Khoo et al. 2015 Oncotarget

Causes of Heterogeneity
Tumors: A Case Study
1. Genetic Mutations
- Variation in the form of acquired Genome instability
mutations • High frequency of mutations
- Stochastic/Unpredictable genomic
• Include changes in nucleic acid
instability, e.g. increased replication
errors
sequences, chromosomal
- Selection by therapeutics rearrangements or aneuploidy.
• Central to carcinogenesis and factor in
some neurodegenerative diseases

http://www.md-health.com/images/10422517/image-1.jpg
Khoo et al. 2015 Oncotarget

1. DNA replication Errors

2. DNA Damage
3. DNA repair deficiencies
4. Chromosomal instability
 Causes of Heterogeneity
Tumors: A Case Study
1. Genetic Mutations
- Variation in the form of acquired Genome instability
mutations • High frequency of mutations
- Stochastic/Unpredictable genomic
• Include changes in nucleic acid
instability, e.g. increased replication
errors
sequences, chromosomal
- Selection by therapeutics rearrangements or aneuploidy.
• Central to carcinogenesis and factor in
some neurodegenerative diseases

http://www.md-health.com/images/10422517/image-1.jpg
Khoo et al. 2015 Oncotarget

Causes of Heterogeneity Causes of Heterogeneity

Tumors: A Case Study Tumors: A Case Study
CCND1 FGFR1 MYC
1. Genetic Mutations 1. Genetic Mutations
- Variation in the form of acquired Selection - Variation in the form of acquired
mutations • For resistance or tolerance mutations
- Stochastic/Unpredictable genomic - Stochastic/Unpredictable genomic
instability, e.g. increased replication instability, e.g. increased replication
errors errors
- Selection by therapeutics - Selection by therapeutics
TOP2A HER2 ZNF217

http://www.md-health.com/images/10422517/image-1.jpg http://www.md-health.com/images/10422517/image-1.jpg
Khoo et al. 2015 Oncotarget Khoo et al. 2015 Oncotarget
 Causes of Heterogeneity
Tumors: A Case Study

2. Epigenetics
- non-genetic influences on
gene expression
- does not involve changes to
the underlying DNA sequence

Causes of Heterogeneity Causes of Heterogeneity

Tumors: A Case Study Tumors: A Case Study

2. Epigenetics DNA methylation 2. Epigenetics DNA methylation

- non-genetic influences on • Process by which methyl groups are • Process by which methyl groups are
gene expression added to the DNA molecule. added to the DNA molecule.
- does not involve changes to • Can change the activity of a DNA • Can change the activity of a DNA
the underlying DNA segment without changing the sequence segment without changing the sequence
sequence • When located in a gene promoter, • When located in a gene promoter,
typically acts to repress gene typically acts to repress gene
Actual factors: DNA transcription. transcription.
methylation, gene silencing
and histone modification
 Gene silencing by miRNA

Causes of Heterogeneity
Tumors: A Case Study

2. Epigenetics Gene silencing

- non-genetic influences on • Regulation of gene expression in a cell to
gene expression prevent the expression of a certain gene.
- does not involve changes to • Can occur during either transcription or
the underlying DNA translation
sequence • Often used in research.

Actual factors: DNA

methylation, gene silencing
and histone modification

Histone Modification

Causes of Heterogeneity
Tumors: A Case Study

2. Epigenetics Histone Modification

- non-genetic influences on • A covalent post-translational
gene expression modification (PTM) to histone proteins
- does not involve changes to • Includes methylation,
the underlying DNA phosphorylation, acetylation,
sequence ubiquitylation, and sumoylation.
• Can impact gene expression by
Actual factors: DNA altering chromatin structure or
methylation, gene silencing and recruiting histone modifiers.
histone modification
 Causes of Heterogeneity Causes of Heterogeneity

3. Mechanotransduction
3. Mechanotransduction

Mechanotransduction refers to the processes through

which cells sense and respond to mechanical stimuli by
converting them to biochemical signals that elicit specific
cellular responses.

Causes of Heterogeneity Causes of Heterogeneity

3. Mechanotransduction 3. Mechanotransduction

Soft

Stiff

Mechanobiology Institute

Li et al. 2015 Scientific Reports

 Causes of Heterogeneity Epithelial to mesenchymal transition
Tumors: A Case Study
3. Mechanotransduction

Cell mechanosensing
Metastasis
Phenotypic changes

Heterogeneity Lower patient survival rate/higher drug resistance

E M

Epithelial to
Tumour invasion and
Matrix stiffness mesenchymal
metastasis
transition (EMT)
Khoo et al. Oncotarget 2015

Epithelial to mesenchymal transition

Epithelial to mesenchymal transition

• A biologic process that allows an epithelial cell, which normally interacts with
basement membrane via its basal surface, to undergo multiple biochemical
changes that enable it to assume a mesenchymal cell phenotype

Includes
• Enhanced migratory capacity,
• Invasiveness,
• Elevated resistance to apoptosis,
• Greatly increased production of ECM components .

• Completion of an EMT is signaled by the degradation of underlying basement

membrane and the formation of a mesenchymal cell that can migrate away from
the epithelial layer in which it originated.

https://link.springer.com/chapter/10.1007/978-1-4939-3363-1_8
 Epithelial to mesenchymal transition

Epithelial to mesenchymal transition

Causes of Heterogeneity Causes of Heterogeneity

Tumors: A Case Study Tumors: A Case Study
4. Tumour microenvironment 4. Tumour microenvironment

anti tumorigenic macrcophage: M1 polarized macrophage

pro-tumorigenic tumour associated macrophage: M2-polarized macrophage

A 3D structure provides a gradient of conditions, leading to heterogeneity

 Causes of Heterogeneity Causes of Heterogeneity
Tumors: A Case Study Tumors: A Case Study
4. Tumour microenvironment 4. Tumour microenvironment

http://wirtzlab.johnshopkins.edu/research/tumor-microenvironment/

What Can We Do?

Causes of Heterogeneity - A Recap

1.Genetic Mutations
2.Epigenetics
3.Mechanotransduction
4.Tumour microenvironment
Today: What can be done? – the 1. Split it up

engineering approach
1. Split it up 2. Capitalise on the odds

Single cell Personalised

analysis treatment

1. Split it up 1. Split it up

Breaking it down Breaking it down

Single cell analysis (SCA) Single cell analysis (SCA) - FACs

Khoo et al. 2015 IJC

http://gecf.epfl.ch/
Assaf Rotem
 1. Split it up 1. Split it up

Breaking it down Breaking it down

Single cell analysis (SCA) - FACs Single cell analysis (SCA) – manual/auto pipetting

1. Split it up
1. Split it up
Breaking it down
Breaking it down Single cell analysis (SCA)
Single cell analysis (SCA) - Microdevices

https://www.nature.com/articles/nature14966
 1. Split it up
2. Capitalising on the odd
Breaking it down
Single cell analysis (SCA) Harnessing differences for management
strategies
Sorting for detecting and disease monitoring - isolating the odd ones – by size (differences in
migration patterns)
The problem:
1. Cell-based analysis • Single biopsy specimens of primary tumours performed for diagnostic purposes,
2. Nucleic acid-based may not fully represent a genetically diverse malignancy with multiple metastatic
analysis sites,
3. Protein-based analysis • Sequencing techniques may not be sufficiently sensitive to detect low frequency
4. Metabolite-based events in tumour subclones.
analysis • Clinical practice is guided by the molecular analysis of primary tumours and we
assume that all primary tumour characteristics are carried over to metastases later
https://onlinelibrary.wiley.co in the disease course.
m/doi/full/10.1002/ijc.30006 • Potentially important molecular changes will be missed if repeat biopsies are not
performed during the evolution of the disease.
• Anticancer treatments may cause selection pressures and influence the complex
mutational landscape within a tumour

What can be done? – the What can be done? – the

biochemical approach biochemical approach
Sorting for detecting and disease monitoring - isolating the odd ones – by size (differences in
• Serial characterization of genetic variants in plasma samples migration patterns)

has the potential to provide information on spatial and temporal • Longitudinal analyses of tumour evolution through the
heterogeneity on a scale that cannot easily be achieved through disease course.
analyses of tumour biopsy samples alone • But multiple tumour biopsies is ethically and clinically
• Multiregion sampling and single-cell sequencing are all challenging
emerging informative platforms that have the potential to enable
decoding of complex clonal relationships at a high level of • New approaches, capitalising on liquid biopsy are essential
resolution
• Combinatorial approaches that pair therapies targeting the
predominant, drug-sensitive population of clones in addition to
the various subsets of drug-resistant and drug-tolerant cells seem
likely to induce the most-durable responses
 2. Capitalising on the odd 2. Capitalising on the odd

Harnessing differences for management Harnessing differences for management

strategies strategies
Sorting for detecting and disease monitoring - isolating the odd ones – by biomarker expression (e.g. Sorting for detecting and disease monitoring - isolating the odd ones – by size (varied distribution
CD44, CK, EpCAM) along a curvilinear channel under inertial force)

Target cell outlet Waste outlet

Khoo et al. 2015 PLOS One

Clearbridge

2. Capitalising on the odd Rare cell analysis and capture – limitations?

Harnessing differences for management
strategies • Have to be carefully studied with rigorous computation,
Sorting for detecting and disease monitoring - isolating the odd ones – by size (differences in which is imperative to distinguish pre-existing genetic
migration patterns)
alterations from amplification errors.
• Multiple independent samples. biasness and errors,
especially in highly heterogeneous samples,
• Since it is technically impossible to eradicate sampling
noise due to the low amount of sample material, additional
steps may be required to distinguish noise from low
prevalence signals. These may be carried out with molecular
fluorescence in situ hybridization (FISH) or unique molecular
identifiers.
• Noise structure from single cell sequencing can also be
determined to differentiate sampling noise from biological
signals.
 2. Capitalising on the odd 2. Capitalising on the odd

Harnessing differences for management Harnessing differences for management

strategies strategies
Personalised targeted therapy Personalised targeted therapy

• Therapy targeting a mutation present in only a fraction of

tumor cells would be expected to affect only that subclone,
leading to limited clinical benefit.
• At worst, targeted therapy might have a paradoxically
stimulatory effect on the subclones lacking the relevant
mutation.
• Important to enumerate the extent of clonal heterogeneity in
patients being evaluated for targeted therapy and to interpret
the results of subsequent therapy in light of such genetic
heterogeneity.

2. Capitalising on the odd

Harnessing differences for management

strategies
Personalised targeted therapy

• Effective targeted therapy will require either drug

combinations targeting distinct subclones or,
• Deployment of targeted therapies only in patients for whom
the drug target is entirely clonal.
• Monitoring over time - patient risk factors, patient response,
and patient adherence ( adaptive treatment strategy concept
used in substance abuse)