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Applying genomic and proteomic microarray technology
in drug discovery 2nd ed Edition Matson Digital Instant
Download
Author(s): Matson, Robert S
ISBN(s): 9781439855645, 1439855641
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Year: 2013
Language: english
 Second Edition

Applying
Genomic and Proteomic
Microarray Technology
in Drug Discovery
Robert S. Matson
 Second Edition

Applying
Genomic and Proteomic
Microarray Technology
in Drug Discovery
 Second Edition

Applying
Genomic and Proteomic
Microarray Technology
in Drug Discovery

Robert S. Matson

Boca Raton London New York

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Taylor & Francis Group, an informa business
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 Dedication

To my family—Jeanne, Erik, and Jacqueline.

© 2010 Taylor & Francis Group, LLC

Contents
Preface.................................................................................................................... xiii
The Author................................................................................................................ xv

Chapter 1 Omics and Microarrays Revisited......................................................... 1

Introduction........................................................................................... 1
The Microarray Format.........................................................................3
Terms and Definitions...................................................................... 3
General Utility.................................................................................. 6
The Biomedical Testing Continuum............................................6
Biotech Sector Trends Revisited.......................................................7
The Omic Era........................................................................................ 8
The Role for Gene Expression Microarrays in Drug Discovery......... 12
Toxicogenomic Applications.......................................................... 15
Proteomics Today—The Great Challenge........................................... 16
The Potential Role for Protein Microarrays in Drug Discovery......... 17
Critical Issues with Protein Microarrays........................................ 18
Stability and Performance......................................................... 18
Content.......................................................................................20
Detection....................................................................................20
Micro-ELISA Formats............................................................... 21
Protein Profiling Formats.......................................................... 22
Near-Term Biomedical Applications.............................................. 22
Cytokines................................................................................... 22
Autoimmune Diseases and Allergy........................................... 23
Future Medicine—Pharmacoproteomics or Pharmacogenomics?.....24
References........................................................................................... 27

Chapter 2 Commercial Microarrays.................................................................... 31

Introduction......................................................................................... 31
In Situ DNA Arrays............................................................................. 31
Ex Situ or Spotted DNA Arrays.......................................................... 39
Content for DNA Microarrays........................................................40
Suppliers of DNA Microarrays....................................................... 41
Comparison of Commercial DNA Microarrays.................................. 41
Commercial Protein Arrays................................................................44
Content Providers........................................................................... 45
An Open Platform Approach..........................................................46
A2 Multiplex ELISA System......................................................46
Three-Dimensional (3D) and Four-Dimensional (4D) Chips.............. 48

vii
© 2010 Taylor & Francis Group, LLC
viii Contents

Flow-Thru Biochips............................................................................. 49
Electronic Biochips............................................................................. 51
References........................................................................................... 54

Chapter 3 Supports and Surface Chemistries...................................................... 57

Introduction......................................................................................... 57
Substrates............................................................................................ 57
Membrane Substrates..................................................................... 58
Glass Substrates.............................................................................. 61
Covalent Attachment................................................................. 61
Adsorptive Attachment..............................................................64
Plastic Substrates............................................................................ 68
Physical Features................................................................................. 71
Hydrogels........................................................................................ 71
Surface Chemistries............................................................................ 72
Linkers............................................................................................ 72
Reactive Groups............................................................................. 81
Preparation of Glass Substrates for Derivatization........................ 85
Variation in the Performance of Glass Slide-Based Antibody
Microarrays......................................................................................... 88
Comparison of Different Surface Chemistries for the
Immobilization of Auto-Antigens.......................................................90
Click Chemistry as an Immobilization Strategy................................. 91
Oxygen Plasma-Mediated Modification of DVD-R Disks for
Tethering of Oligonucleotides............................................................. 93
Construction of Lipid Bilayer Microarrays.........................................96
Summary.............................................................................................97
References........................................................................................... 98

Chapter 4 Arraying Processes............................................................................ 103

Introduction....................................................................................... 103
Creating Spotted Microarrays........................................................... 104
Substrates...................................................................................... 104
Print Buffer................................................................................... 105
The Printing Environment............................................................ 106
Microarray Printing Mechanisms..................................................... 106
Microarray Pins................................................................................. 113
Other Approaches.............................................................................. 115
Printer Performance..................................................................... 121
Pin Performance........................................................................... 121
Microarray Design........................................................................ 125
Setting Up the Print Run................................................................... 128
Printing Parameters...................................................................... 130
Preparing the Probe Ink............................................................... 133

© 2010 Taylor & Francis Group, LLC

Contents ix

Optimization of Probe Concentration.......................................... 133

Protocols for Printing Nucleic Acids................................................. 134
cDNA Microarray......................................................................... 134
Oligonucleotides........................................................................... 135
Use of DMSO............................................................................... 136
Use of Betaine.............................................................................. 136
Use of Polyvinyl Alcohol.............................................................. 138
Evaporation................................................................................... 138
Print Quality Assessment............................................................. 141
Backgrounds................................................................................. 144
Protocols for Printing Proteins.......................................................... 146
Antibody Arrays........................................................................... 148
Newer Methods for Printing.............................................................. 153
Acoustic Dispensing..................................................................... 153
Flexographic Printing................................................................... 155
Microarray-to-Microarray Slide Snapping Transfer................ 157
Micro-Contact (µCP) Printing..................................................... 158
Creation of Peptide Nucleic Acid Microarrays by µCP........... 161
Covalent Subtractive Print by µCP.......................................... 163
References......................................................................................... 164

Chapter 5 Gene Expression: Microarray-Based Applications........................... 169

Introduction....................................................................................... 169
Applications Demonstrating DNA Microarray Utility..................... 169
Gene Expression........................................................................... 169
Biomedical Research Applications................................................... 180
Drug Discovery............................................................................ 180
Drug Toxicity........................................................................... 182
Cancer...................................................................................... 185
Infectious Disease.................................................................... 197
Other Disease States................................................................200
Micro-RNA....................................................................................... 203
The Utility of Gene Expression Microarrays in Micro-RNA
Analysis........................................................................................204
The Nature of Platform-to-Platform Disparity.............................206
Array-Based Comparative Genomic Hybridization.......................... 210
Karyotype, Fluorescence in situ Hybridization (FISH),
Comparative Genomic Hybridization (CGH), or Array-CGH?...... 211
References......................................................................................... 213

Chapter 6 Protein Microarray Applications....................................................... 217

Introduction....................................................................................... 217
Spot Theory.................................................................................. 217
Applications Demonstrating Protein Microarray Utility.................. 223

© 2010 Taylor & Francis Group, LLC

x Contents

Microtiter-Based Antibody Arrays...............................................224

Membranes................................................................................... 225
Glass Slides................................................................................... 228
Measuring Microarray Performance................................................. 235
Sensitivity and Dynamic Range................................................... 235
Other Microarray Formats Useful for Proteomic Applications........ 242
mRNA-Protein Fusions................................................................ 242
PISA..............................................................................................244
Aptamers......................................................................................246
Universal Protein Array............................................................... 249
Peptide Arrays for Antibody Detection........................................ 254
Phage-Display Antibody Selection............................................... 255
Protein Kinase Microarray........................................................... 255
Protein Microarrays Useful in Auto-Antibody Screening........... 256
Dual Labeling of Targets for Increased Sensitivity and
Specificity...................................................................................... 258
The Depletion of Highly Abundant Proteins from Serum
Deemed Unnecessary........................................................................ 259
Competitive ELISA by Protein Microarray...................................... 259
The Issue of Cross-Reactivity in a Protein Microarray Sandwich
ELISA................................................................................................260
References......................................................................................... 263

Chapter 7 Multiplex Assays............................................................................... 267

Introduction....................................................................................... 267
Benefit........................................................................................... 267
Definition...................................................................................... 267
Multiplex Polymerase Chain Reaction (PCR)................................... 271
Multiplex PCR Compared to DNA Microarray........................... 272
Multiplex Lateral Flow...................................................................... 272
Nucleic Acid Lateral Flow Immunoassay (NALFIA).................. 274
Multiple Test Lines....................................................................... 276
Multiplex Bead-Based Assays........................................................... 276
Multiplex Bead-Based Assay Compared to the ELISA............... 277
Multiplex Bead-Based Herpes Simplex Virus (HSV) Assay
Compares Favorably with Serological Methods........................... 279
False Readings.............................................................................. 281
Observed Differences between ELISA and Bead-Based
Multiplex....................................................................................... 281
Combined Multiplex PCR and Multiplex Bead-Based Assay...... 282
Multiplex Microarrays....................................................................... 283
Multiplex Microarray Plate Assay Compared to the ELISA........284
A Lack of Standardization........................................................... 286
Label Free Detection of a Multiplex Small Molecule
Microarray.................................................................................... 286

© 2010 Taylor & Francis Group, LLC

Contents xi

Adoption of Multiplex Assays........................................................... 288

Trends........................................................................................... 290
References......................................................................................... 291

© 2010 Taylor & Francis Group, LLC

Preface
Microarrays remain an invaluable tool for omics-based research in the life sciences,
for drug discovery, clinical research, and diagnostic applications worldwide. They
are used for gene expression analysis, the assessment of genomic mutation, and mul-
tiplex immunoassays. We can create patterns of macromolecules such as enzymes
and living cells with array-based technologies. They have a future in companion
diagnostics and personalized medicine. The purpose of this book is to introduce
microarrays into your future.
In this second edition I have sought to add new information about the construc-
tion and use of microarrays while preserving the important earlier works that dem-
onstrated key concepts. The commercial landscape has dramatically changed over
the past few years so it has been necessary to make an accounting of these changes
and reflect upon the contributions that these companies provided in the advancement
of array technology. There are also new surface chemistries and immobilization
strategies that have emerged for tethering and patterning of nucleic acids, proteins,
carbohydrates, cells, and other ligands. Protein microarrays are used extensively.
Thus, new applications as well as the performance of antibody arrays have been
reviewed. A new chapter, “Multiplex Assays,” has been added to examine the devel-
opment and application of arrays across diverse platforms. We discuss applications
for qPCR, multiplex lateral flow, multiplex bead assays, as well as microarrays; and
offer platform-to-platform comparisons.
There is little doubt that these omics tools have changed our approach to problem
solving. We are said to be omics-driven rather than hypothesis-driven, which is a
kind of “shoot first and ask questions later” approach to discovery. After all of these
years, I still view microarrays as an enormous opportunity.

Robert S. Matson, PhD, FACB

Orange, California

xiii
© 2010 Taylor & Francis Group, LLC
The Author
Robert S. Matson is president and co-founder of QuantiScientifics, LLC. The
company is involved in the commercialization of multiplexed assays for life sci-
ence, clinical research, and in vitro diagnostics. Previously, Matson was involved
in the research and development of microarray technologies, detection chem-
istries, as well as point-of-care devices for more than seventeen years while at
Beckman Coulter, Inc. He participated in the National Institute of Standards and
Technology’s (NIST) Advanced Technology Program sponsored Genosensor
Consortium and collaborated with Sir Edwin Southern on the development of an
in situ oligonucleotide array synthesis platform for the corporation. Other work
included development of the A2 MicroArray System, a microplate-based array
platform for multiplexed micro-ELISA, which QuantiScientifics recently licensed
for commercialization.
Matson has been granted twelve U.S. patents and six European patents on nucleic
acid and protein microarray technology. He was inducted into Beckman Coulter’s
Inventors Hall of Fame in 2006 and was elected a Fellow of the National Academy
of Clinical Biochemistry in 2008. He previously served in several technical man-
agement roles including Research and Development (R&D) Director, BioProbe
International; R&D Director, Costar-Nuclepore; and Chemistry R&D Group Leader
at BioRad Laboratories. Matson earned his PhD in biochemistry from Wayne State
University. Following postdoctoral studies at the UCLA Medical School, he served
as a principal investigator with the Veterans Administration Medical Center and
as adjunct professor of biological chemistry at the University of California-Davis
Medical School. He has also held a faculty lectureship at USC’s Department of
Chemistry and was assistant professor of chemistry at the University of Southern
Maine, Portland. He is the author of Applying Genomic and Proteomic Microarray
Technology in Drug Discovery (CRC Press, 2005) and Microarray Methods and
Protocols (CRC Press, 2009).

xv
© 2010 Taylor & Francis Group, LLC
 1 Omics and Microarrays
Revisited
These biomarker patterns will provide more individualized information, which
will then provide support to clinicians and selection of optimal therapies.
Thomas O. Joos*

INTRODUCTION
In the previous edition of this book (2005) we spoke of an “omics-driven” paradigm
shift toward quantitative biology in which the microarray played a key role. Indeed,
we have witnessed the continued advancement of DNA-array-based platforms as
powerful gene expression analysis tools for biomarker discovery. Protein arrays con-
tinue to play a role in biomarker discovery and validation but are also in demand for
both clinical research and clinical diagnostic applications (Yu et al., 2010). Clearly,
much debate remains over the clinical role for biomarkers, as well as the validation
and regulation of derived multianalyte tests and multivariate index assays (Amur
et al., 2008; Tahara et al., 2009; Boja et al., 2011; Smith, 2011; Dimond, 2012;
Wellhausen & Seitz, 2012).
Nevertheless, microarrays are now transitioning for use in companion diagnos-
tics. For example, Merck and Roche began collaboration in 2011 to develop the
Affymetrix’s AmpliChip p53 for companion diagnostics. Roche Diagnostics will
validate and standardize tests for Merck’s use in qualifying patients in clinical oncol-
ogy trials. Clinical proteomics has emerged as a discipline; the first protein-based
multivariate index assay, the OVA1 test for ovarian cancer, received U.S. Food and
Drug Administration (FDA) approval in 2009.
We have also witnessed the emergence of other omics-driven research employ-
ing microarray technologies. The glycomics field (see Consortium for Functional
Glycomics [CFG] at www.functionalglycomics.org) has adapted microarrays for
the screening of glycan-protein interactions. For example, Smith and co-workers
at Emory University (Atlanta, Georgia) have developed “shotgun glycomics” as a
means to identify and characterize the functionality of glycans (Song et al., 2011).
Oligosaccharide (glycan) arrays have been used in identifying immunogenic car-
bohydrate moieties on the pathogen, Bacillus anthracis exosporium (Wong et
al., 2007). The Stanford University group has been active in the development of
photochemical micropatterning of carbohydrates (Carroll et al., 2006) and in the
photogeneration of epitope-specific glycan arrays using phthalimide terminated

* Protein Microarrays for Personalized Medicine, Clin Chem 56(3), 376–387, 2010.

1
© 2010 Taylor & Francis Group, LLC
2 Genomic and Proteonomic Microarray Technology in Drug Discovery

self-assembled monolayers (PAMs) on photo-reactive surfaces (Wang et al., 2002).

Godula and Bertozzi (2010) describe a poly(acryloyl hydrazide) (PAH) scaffold for
the rapid immobilization of reducing glycans. They used biotin terminated PAH
conjugated glycans to assemble a microarray on streptavidin-coated glass slides.
The utility of glycan microarrays for elucidating the glycome was recently reviewed
in the Annual Review in Biochemistry (Rillahan & Paulson, 2011). For additional
information regarding the preparation and use of carbohydrate arrays as well as lec-
tin microarrays, you are encouraged to review Microarray Methods and Protocols
(Matson, 2009).
Metabolomics seeks to understand the functionality (pathways, networks) and
biochemical status of metabolites within the metabolome, an accounting of approxi-
mately 1800 biochemicals with molecular masses of under 1500 daltons. These
would include small molecules and compounds such as peptides, amino acids, sug-
ars, antioxidants, and lipids. Psychogios et al. (2011) applied a number of metabo-
lomic analytical tools to begin characterization of the serum metabolome (see the
Serum Metabolome Database [SMDB] Web site at www.serummetabolome.ca). The
SMDB has assembled at least 4500 biochemicals present in serum and plasma with
information regarding concentration levels in normal and disease states. They used
NMR (nuclear magnetic resonance) spectroscopy, along with various hyphenated
mass spectroscopy (MS) methods: GC-MS (gas chromatographic MS), LC-MS (liq-
uid chromatographic MS), TLC (thin-layer chromatographic)/GC-MS, as well as
tandem mass spectroscopy (MS/MS) such as UPLC- (ultra-high-performance LC)
MS/MS, and direct flow injection (DFI) MS/MS in their quest to identify, quantify,
and validate plasma and serum metabolites. Progress in microbial metabolomics has
been reviewed by Jane Tang from the Center for National Security and Intelligence
(Tang, 2011). The author points out that studies aimed at an understanding of the gut
metabolome are particularly important because microbes are intimately involved in
drug detoxification, xenobiotics, allergy, and human disease. What is not used to any
appreciable extent in metabolomics research at this time is microarray technology.
However, Borgan et al. (2010) applied both gene expression microarray (transcrip-
tome) analysis and magnetic resonance spectroscopy (metabolome) for investiga-
tions that resulted in a more refined subclassification of breast cancers.
Transcriptomics involves characterizing the transcriptome, essentially determin-
ing the functionality and relationship of some 100,000 mRNA transcripts being
processed within cells of an organism. DNA microarrays have been extensively
used for gene expression studies and the temporal analysis of transcripts. In their
review, Malone and Oliver (2011) compared the virtues of gene expression micro-
arrays and deep sequencing of RNA based upon RNA-Seq analysis. mRNA-Seq
involves the use of fragment libraries of RNA that are then converted to cDNA by
reverse transcription. This is followed by an adaptor ligation step and sizing for use
on next-generation sequencing platforms. While RNA-Seq may be more applicable
for discovery, the authors found both microarrays and deep sequencing to be use-
ful: “We conclude that microarrays remain useful and accurate tools for measuring
expression levels, and RNA-Seq complements and extends microarray measure-
ments” (p. 1).

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 3

THE MICROARRAY FORMAT

The microarray, in its most elementary form, is but a collection of small spots of biologi-
cal capture agents (DNA, antibody, carbohydrate, etc.) organized on a planar substrate
such as a glass microscope slide. With this tool we are free to move beyond our rather
myopic views of the cell in favor of a more global assessment of the cellular process.
Moreover, the microarray provides us with digital information that we can assemble
and then use to quantify biological events and relationships on a scale that was unimagi-
nable a few decades ago. Finally, the microarray is a parallel processor providing the
researcher with a rapid response to the biological query. In fact, so much data can be
obtained in such a short time that it can be overwhelming, most often requiring the aid of
sophisticated bioinformatics analysis software. Thus, the microarray has become a for-
midable instrument by which to quantify biology. The purpose of this book is to assess
progress on the utility of microarray technology to solve important biological problems.

Terms and Definitions

Array technology finds its origins within molecular biology, a scientific field of study
notoriously irreverent to the rules governing systematic nomenclature (Figure 1.1).
Moreover, microarray applications have crossed over into other scientific fields, each
with its own unique terminology. Below are some general terms that apply to the
microarray field. Other terms and further elaboration with illustration can be found,
for example, in the concise A-to-Z guide, Microchips: The Illustrated Hitchhiker’s
Guide to Analytical Microchips (Kricka, 2002). A more recent review traces the
evolution of arrays from muffin pans and test tube racks (Kricka et al., 2010).
The DNA array has been categorized into different formats based upon what is
immobilized to the surface (also known as the solid-phase, substrate, or chip) and
what is captured out of the sample solution. Definitions change depending upon the
format. For the classic Southern dot blot the sample was first spotted down on the
surface, cross-linked, and then bathed with a radiolabeled oligonucleotide under
hybridization (complementary nucleic acid strand base-pairing) conditions to detect
the presence of a particular sequence within the sample. This was called probing.
The oligonucleotide was the probe, and the DNA in the sample that was immobilized
onto the surface became known as the target. Subsequently the Southern dot blot
hybridization process was classified as Format I (or the forward blot) and was associ-
ated primarily with membrane blots. The invention of the reverse blot (Format II)
led to some confusion in the early days of microarrays. Here many different probes
are immobilized onto the substrate and the sample (target) labeled for hybridization
(Figure 1.2). There are additional formats that will be described later on. In most
instances, however, we will be discussing Format II microarrays for both nucleic acid
and protein capture. The generally accepted meaning of the probe is as the solid-
phase capture agent (Figure 1.3). In drug discovery, the target is usually the protein
(or protein complex) that chemically interacts with a compound (drug candidate).
There are also terms related to the anatomy of the microarray (Figure 1.4). The
probes are immobilized on the substrate at discrete (x, y) locations or spatially
addressable sites. The probe spots (measured in microns- diameters for circular

© 2010 Taylor & Francis Group, LLC

4 Genomic and Proteonomic Microarray Technology in Drug Discovery

Southern Dot Blot Filter

Gridded cDNA Filter Gridded ASO Filter

SBH Chips

Slide cDNA Microarray

Slide Oligo Microarray

FIGURE 1.1 Origin of the microarray.

Format I: Format II:

Southern (forward) blot Reverse blot
classical approach, 1960s-present modern era, 1988-present
e.g., nitrocellulose membrane e.g., biochips
hybridization “microarray”
“macroarray”

FIGURE 1.2 Hybridization blot formats.

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 5

Target = the analyte in Array = collection of

the sample that you wish probes arranged on a
to identify (pcr amplicon; surface or substrate
cDNA; mRNA — usually
labeled) Features or Elements
refers to the number of
spots of probes present
on the array

Probe = known sequence

attached to substrate
Hybridization = the which captures the target
process by which complementary or mismatch
probe and target bind; sequence from solution
thermodynamic rules (oligonucleotide; cDNA)

Substrate = the solid-phase surface

(glass, silica, plastic, acrylamide, gold)

FIGURE 1.3 Microarray anatomy.

t
taa
gt ttct
ggtta

Target
AAGAGCGGCTCCACCACT

Elements or features
Probe (spots, 75–150 micron dia.)

Center-to-center spacing
Substrate (150–300 microns)

FIGURE 1.4 Microarray terms.

© 2010 Taylor & Francis Group, LLC

6 Genomic and Proteonomic Microarray Technology in Drug Discovery

spotted arrays; or the side of a square for in situ arrays) are often referred to as
features or elements of the array. Thus, an array containing 10,000 features would
have 10,000 probes arranged as an array on the substrate. Typical spotted arrays
would have 100 to 150 micron (µm) diameter features, while the photo-lithograph-
ically prepared in situ arrays may have features on the order of 2 to 20 µm. The
separation between elements is usually measured in terms of a center-to-center
distance, spacing, or pitch. That is, for a printed array, two adjacent spots in the
array (e.g., each at 100 micron spot diameter) may have a center-to-center distance
of 150 microns. Or, the spots would be separated by 50 microns from their edge.
The number of spots per square centimeter usually defines the spot density. As
an example, Affymetix manufacturing arrays at >280,000 elements per 1.28 cm ×
1.28 cm chip would have a spot density on a chip of 170,000 elements/cm2. We can
also classify arrays in terms of high, medium, and low probe density. There may be
some argument regarding the precise boundary limits distinguishing microarrays
on a density scale. Nevertheless, a high density array would contain >10,000 probes/
cm2; medium density, 1000 to 10,000 probes/cm2; and low density, <1000 probes/
cm2. There are also definitions for arrays based upon whether or not they are macro-
and microarrays. A macroarray could be regarded as having larger and fewer spots
than a microarray. For example, the Southern blot on a standard sized membrane (~
8 cm × 12 cm; nylon or nitrocellulose) with spot diameters of 500 microns would be
considered a macroarray by most researchers. However, macroarrays can have thou-
sands of printed spots per membrane and thus functionally perform at a level similar
to that of the slide microarray.
The number of probe molecules per square millimeter defines the spot’s probe
density. Probes within an element or spot could have densities on the order of 109
to ~1012 molecules/mm2 depending upon the molecular size of the nucleic acid (e.g.,
short oligonucleotide versus cDNA).

General Utility
As we will soon discover, microarray-based technologies have found utility in a
number of fields. While DNA arrays are the most technically mature and have the
broadest application portfolio, we have witnessed the ever-increasing generation of
new kinds of probe arrays: antibody, antigen, enzyme, aptamer, carbohydrate, tissue,
cell, and small molecule microarrays. The list undoubtedly will continue to expand.
We can also describe microarrays in terms of prognostic, diagnostic, and predictive
roles. A few examples that examine these applications are provided.

The Biomedical Testing Continuum

The primary focus for microarrays has been for biomedical-related analysis.
Biomedical testing can be divided into four major areas: discovery testing, clini-
cal trial testing, specialized testing, and patient care testing (Figure 1.5). Most
emerging technologies are likely to have been first invented or developed during
the discovery process. In the area of biomedical testing, new technologies will pass
from research (academic, biomedical, pharmaceutical) into specialized testing are-
nas such as core facilities or outsourced (esoteric testing) laboratories, and hopefully

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 7

Hospital/POC
Patient Care Clinics/M.D. offices
Testing Blood Banks
Biochip Migration Patient Self-Tests
within
Biomedical Testing
Continuum

Discovery Testing
Specialized University Res. Labs
Testing NIH other Gov. labs
BioPharma/Biotech
Core Facilities
Commercial Labs
BioPharma
Diagnostic Labs

Clinical Trials Testing

University/Med. Ctr.
BioPharma/Biotech
CRO

FIGURE 1.5 Biochip migration.

be adopted from these into the mainstream of diagnostics. Array technology finds
its roots in academic circles as a means to sequence genomes. The reverse blot was
originally constructed at a diagnostic research center as a new nucleic acid assay
format for the simultaneous detection of infectious agents.
In the first edition (2005) we observed limited success at moving array technol-
ogy directly into point of care (POC) or centralized laboratory testing. However, that
has changed (see Chapter 7, “Multiplex Assays”), and you will now find microarray
technology being adapted for POC in the form of biochips and lateral flow strips.
Bead-based multiplex assays (or “fluidic” arrays) are in use for clinical research and
are anticipated to migrate into in vitro diagnostics (IVD) and animal health diagnos-
tics in the near future.

Biotech Sector Trends Revisited

I previously characterized the application of array technology as riding upon
the “omic” wave (Figure 1.6). There is no doubt that gene expression and geno-
typing microarray technology have evolved into mature products (see Chapter 2,
“Commercial Microarrays”). These have benefited greatly from the availability of the
human genome sequence database as well as other sequenced genomes (Genomics
I). Whole genome chips are offered by numerous vendors. However, next generation

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8 Genomic and Proteonomic Microarray Technology in Drug Discovery

NGS
Genomics II

c
s Spe
micro-RNA

Mas
Cell array
aCGH
SNP II Bioinformatics
Proteomics
Antibody/Antigen array
Tissue Array
Genomics I
G i expression
Gene
SNPs
Mutation
CF, HLA SBH

Transcriptomics
Metabolomics
Glycomics

FIGURE 1.6 Microarray technology waves.

sequencing (NGS) has the potential to displace microarrays in certain fields. For a
review of NGS technologies see Metzker (2010).
Protein microarrays, both planar and bead-based formats, have been widely
adopted for biomarker discovery, biomarker validation, life science research, and
clinical research. Multiplex immunoassays are being evaluated for use in animal
health and human disease diagnostics. However, most proteomic studies continue to
use two-dimensional (2D) gel electrophoresis and mass spectroscopy tools, especially
matrix-assisted laser desorption-mass spectometry (MALD-MS). Improvement in
mass spectroscopy for peptide and protein analysis (Whiteaker, 2010) may challenge
microarrays for clinical use.
A second genomics wave (Genomics II) has emerged with growth in array-based
genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) analy-
sis for genetic diagnosis. Micro-RNA (miRNA) gene expression profiling, espe-
cially in cancer research, is also underway using microarrays (see Chapter 5, “Gene
Expression: Microarray-Based Applications”).
Finally, microarray complexity has increased dramatically over the past decade
requiring advanced statistical treatments, ever increasing our thirst for bioinformat-
ics. Information technology (IT) plays a key role in microarray-based platform vali-
dation (Figure 1.7).

THE OMIC ERA

A major driving force in the advancement of microarrays has been the Human
Genome Project and the development of the genomics field. Oddly enough, the ori-
gins of DNA array technology begin with efforts at sequencing by hybridization

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 9

Market Valuation ($ billions) 10

8
IT

0
1998 2000 2003 2006
Year

Drug discovery Genomics Proteomics

DNA microarray

FIGURE 1.7 Biotech sector market growth.

(SBH) of the human genome. Except for a few private ventures such as the work
at HySeq, the SBH approach was quickly supplanted by dideoxynucleotide dye-
terminated gel-based sequencing. The other early use of DNA arrays was in mutation
screening such as for cystic fibrosis (CF) mutations or in human leukocyte antigen
(HLA) typing. The problem with screening for mutations associated with genetic
disease remains a lack of therapy or cure. Without the availability of treatment there
is very little incentive to produce a diagnostic test. The other issue with mutation
detection is that the prognosis is often a statistical inference. For example, possess-
ing the BRCA1 gene mutation only increases the probability risk or likelihood that
a woman may develop familial breast cancer. The presence of the mutation alone
cannot unequivocally determine whether or not someone will develop breast cancer.
Other factors must be considered, especially in this case the family history regard-
ing the occurrence of breast cancer. Thus, mutation detection based upon microarray
technology has not grown as rapidly as once anticipated.
The breakout opportunity for widespread adoption of array technology came
largely from gene expression studies in which the expression levels from two cell
states (e.g., control versus drug-induced; normal versus diseased) were compared.
So while sequencing efforts defined structural genomics, the gene expression
microarray became the tool for functional genomics. In particular, gene expression
microarrays were found to be well suited as a new kind of differential display tech-
nology applied to the drug discovery process. Here, pharmacogenomics examines
the responsiveness of genes (cellular mRNA levels) to drug candidates. The micro-
array measuring the mRNA levels within the cell’s genome permits identification
of potential targets (albeit, mRNA serving as the surrogate of translated protein)

© 2010 Taylor & Francis Group, LLC

10 Genomic and Proteonomic Microarray Technology in Drug Discovery

Cell Line Library

Select cell type and place in culture

96-well tissue culture

Barcode

Grow cells in presence or absence of drug

Cells +/– drug
Experiment

cDNA Library
Sample
Select Genes
Remove cells
extract nucleic acids
Drug Screen Set
Label RNA/DNA Formulate drugs at specific conc. Compound Library
Hybridize

Make cDNA
microarray Analysis Data Base

Bioinformatics

FIGURE 1.8 Off-target analysis.

or alterations to metabolic pathways, thereby implicating the participation of other

targets. Metabolic alterations may also lead to “off-target effects” that are adverse or
toxic. Therefore, microarrays have been found to be very useful in toxicogenomic
applications. In essence, mRNA profiling using microarrays can potentially reduce
the need for extensive animal model or tissue studies at the onset of the target dis-
covery phase, as well as in the later preclinical phase looking at off-target toxicity
(Figure 1.8).
Gene expression analysis has recognized limitations (Lillie, 1997; Clarke et al.,
2001). The monitoring of transcriptional events serves as an indirect measure of pro-
tein (target) expression. Because proteins can undergo post-translational modifica-
tion leading to subcellular localization and pooling, export, degradation, or complex
formation with other proteins, the correlation between mRNA level and putative
protein is often lost (Figure 1.9).
It is important to consider what both genome- and proteome-based arrays offer
(Figure 1.10). The gene expression microarray monitors relative mRNA abundance
between two cell populations, provided control and sample populations are processed
in the same manner. This is often spoken of as a “snapshot” of gene activity. That
is, unless we also sample these populations, often only a finite view of the changes
in cellular activity between the two sets will be recorded. We may need at times
many snapshots or time course studies. The great advantage of the gene expression
array is that it permits a global analysis of the genome. Later I will be relating how
genome-based approaches provide good information about cell cycles and pathways,

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 11

Gene Expression

hRNA Transcription
Gene
Splice variants
mRNA

Why use proteins?

Ribosomes Apo-protein
Protein Expression
Nascent
protein product Post-translational Further
modification processing
Translation

Localization +

Holo-protein

FIGURE 1.9 Why use proteins?

Genome-based arrays

Relative mRNA abundance

“snapshot” of gene activity
surrogate biomarkers or fingerprints
cell cycle

Proteome-based arrays

Relative protein abundance

end-point expression pattern
gene function
post-translational modifications
+ protein localization biomarkers

FIGURE 1.10 Genome- versus proteome-based arrays.

leading to the discovery of potential surrogate biomarkers. Gene expression microar-

rays remain an important tool for several reasons:

1. Availability of full genome representation

2. Ability to amplify for detection of lower abundant mRNAs
3. Simple capture process based upon hybridization thermodynamics and pri-
mary sequence information

© 2010 Taylor & Francis Group, LLC

12 Genomic and Proteonomic Microarray Technology in Drug Discovery

Obviously for other reasons, such as RNA splice variants and the post-trans-
lational modification of proteins, one cannot rely on mRNA profiling alone.
However, development of a complete “proteome chip” that would provide global
inspection of relative protein abundance has been hindered. There is simply little
means of obtaining a highly significant representation of a proteome (i.e., con-
tent) on any chip. Antibody arrays have been introduced but are of limited util-
ity at this point for proteome-wide applications. Because microarrays are closed
architecture technology platforms, they can only provide information based upon
what is contained on the chip. So, we must first acquire enough protein content
(antibody libraries or mimetic agents such as aptamers) necessary for proteome
discovery work.
Some argue that it is a waste of time monitoring gene expression when ultimately
we desire the end product, the protein target. More likely both are needed to further
grasp at our understanding of cellular events (Clarke et al., 2001). We now have our
list of genes in hand and a few good tools. Not surprisingly we have found biology
to be even more complex than once thought. As John Weinstein suggests, “perhaps
the most important (and least recognized) aspect of biology that makes it difficult to
understand systematically is the fact that biological complexity was not produced by
a watch maker or an engineer” (2002, p. 362).
So, while we can consider the strengths and weaknesses of various technologies
and pursue “omic” approaches, we must also not lose sight that these are tools to aid
in our study. Miller (2002) makes this point:

These novel and significant tools, which are rapidly becoming indispensable, do not
by themselves enlarge the bedrock of basic knowledge that underlies new discoveries.
That foundation remains the detailed understanding of the biological basis of health
and disease.

THE ROLE FOR GENE EXPRESSION

MICROARRAYS IN DRUG DISCOVERY
Fundamental approaches in the drug discovery process have in themselves under-
gone a paradigm shift (Neamati & Barchi, 2002). The pharmaceutical industry
has embraced molecular biology, adopted robotics for high-throughput screening,
and supported those efforts aimed at genome sequencing and SNP identification
as related to disease. Microarray technology, especially gene expression chips, has
been well received in the drug discovery labs of Biopharma. According to Neamati
and Barchi (2002) modern drug discovery can be arranged into five major areas
(Figure 1.11):

1. Target Identification (discovery)

2. Target Validation
3. Lead Identification (hits)
4. Lead Optimization
5. Preclinical Pharmacology/Toxicology

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 13

Drug Discovery
Target II
Clinical
Validation
Leads Preclinical
I III

Start Target Hits

Discovery

FDA
IV

R.O.I.

FINISH

FIGURE 1.11 Drug discovery processes.

In the target discovery process we have witnessed an increased role for genomics.
In fact, as much as 25% of new target identification efforts may now be based upon
genomic approaches. Differential gene expression pattern analysis (control versus
drug response phenotype; normal versus diseased state) developed using the micro-
array chip technology continues to play an important role. High-density microarrays
such as Affymetix’s GeneChip have been found to be very useful in target identi-
fication. Microarrays based upon normalized cDNA libraries have also been suc-
cessfully used in the discovery of novel genes that are potential candidates for drug
targeting (Katsuma, 2001). cDNA arrays have now given way to high-density oligo-
nucleotide chips that represent whole genomes.
The human genome is “roughly” complete as are the sequences of useful
model genomes (e.g., mouse, rat, yeast) nearly complete with other genomes being
sequenced at a rapid pace. The actual number of fully completed genomes is rather
elusive, but counting microbes there are most likely several hundred completed or
nearly completed sequences. Gene expression arrays should continue to serve with
increased capacity provided they are not displaced by next-generation sequencing
technologies. Gene expression profiling of metabolic pathway enzymes can also play
a pivotal role in guiding efforts toward identifying new targets.
While we may be remised in that differential gene expression is but a surrogate
methodology, we do not yet have an equivalent proteomic tool. Albeit, the NAPPA
(nucleic acid programmable protein array) approach (Ramachandran et al., 2008)
could potentially be used for this purpose (Anderson et al., 2011).
Ringer et al. (2000) found pharmacogenomics was rapidly becoming an accepted
route in the later stages of the drug discovery process involving both the preclinical
and clinical phases. A key factor in the acceptance of the pharmacogenomic (and

© 2010 Taylor & Francis Group, LLC

14 Genomic and Proteonomic Microarray Technology in Drug Discovery

pharmacogenetic) approach has been that both drug efficacy and toxicity are well-
correlated to changes in gene expression. Microarrays offer both high throughput
and sensitivity. These attributes are particularly advantageous in reducing the time
and cost in determining drug toxicity during the preclinical stage. During late stage
clinical trials the profiling of an individual’s genetic variation (SNP) correlated to
drug response has been an important screening process. Several microarray-based
SNP “calling” platform technologies (e.g., Affymetrix Genome-Wide Human SNP
Array; Illumina’s Golden Gate SNP Assay) can be used in defining specific poly-
morphisms associated with variable drug responses within individuals and various
populations.
Without a doubt one of the most historic testimonies to the power of the microar-
ray has been in the characterization of cancer cell lines and tumor gene expression.
The National Cancer Institute’s study of cDNA microarray gene expression patterns
across 60 human cancer cell lines (with an activity pattern database on each cell line
individually challenged by over 70,000 compounds) remains the “tour de force” in
microarray-based profiling (Ross et al., 2000). Even more impressive has been the
assembly of the integrated gene expression-molecular pharmacology database for the
NCI60 cell lines (Scherf et al., 2000). And while the NCI60 study had some recog-
nized limitations (limited activity assay data; limited number of arrayed genes; sur-
rogate relationship of cell line to tumor cells), there are those encouraging examples
where the gene expression approach has unraveled mechanisms of drug resistance.
Rew (2001) considered five areas in cancer research applicable for the use of
microarray technologies:

1. Tumor classification—Drug treatment regimes often depend upon tumor

type. The origin of metastatic tumor cells can be difficult to determine by
conventional histopathology. Gene expression profiling may complement
the more traditional methodology where tumors are difficult to classify.
2. Mutation detection—Genetic mutation leading to disease states such as in
familial breast cancer can be detected by arrays. This is provided that suf-
ficient gene probe sequence content is available in order to make a statisti-
cally significant prognosis.
3. Gene copy number—Tumors are known to contain variable numbers of
genes relative to normal tissue. Comparison between the normal versus
tumor gene populations is useful in identifying tumors and their differenti-
ated state.
4. Cancer therapeutics—Different tumor types and the differentiated states
of individual tumors are known to exhibit unique gene expression patterns,
called molecular signatures or fingerprints. The molecular signature may
be useful in tumor phenotype classification.
5. Drug sensitivity—Microarrays are useful in monitoring both on-target
and off-target drug responses. Knowledge of which genes are up- or
down-regulated leads to an understanding of the mechanisms of action
and new treatments.

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 15

In the review by Zanders (2000), a good case is made for the use of gene expres-
sion microarrays to monitor changes in signaling pathway activity. The rationale is
that under environmental stress such as in tumor growth or during an inflammatory
response, signaling pathways are activated or repressed, and these events can be
measured by gene activity by mRNA profiling. That is, “transcription of mRNAs
could be exploited as a ‘surrogate marker’ of signaling pathway activation” (Zanders,
2000, p. 378). Cited examples include studies concerning the expression profiling of
signaling pathway enzymes in yeast such as the mitogen-activated protein kinases
(MAPKs) during times of growth or differentiation or when under stress (Roberts
et al., 2000). Homologous mammalian MAPKs have been reported and are being
investigated as potential drug targets. In the study by Iyer (1999), the use of gene
expression microarrays revealed the induction of genes involved in wound healing
during serum-stimulated human fibroblast growth.

Toxicogenomic Applications
Almost without exception, gene expression is altered during toxicity, as either a direct
or indirect result of toxicant exposure (Nuwaysir et al., 1999).

This early cause-and-effect revelation most certainly promoted the early use of
the gene expression microarray technology at the National Institutes of Health (NIH)
resulting in the creation of the ToxChip cDNA microarray. A toxicant signature
is derived from gene expression relative fold changes between control and treated
cell populations. The ToxChip contained approximately 2000 human gene cDNAs
arranged in various functional categories such as apoptosis, cell-cycle, cytochrome
P-450s, and so on, to detect response to toxic insult. Hodges et al. (2003) were able
to elucidate the mechanism of action for tamoxifen, a drug used in the treatment of
breast cancer, using the ToxChip. Lobenhofer et al. (2002) used the ToxChip v1.0 to
study the mechanism of estrogen-induced proliferation (mitogenesis) using a hor-
mone-responsive human breast cancer cell line.
Even though toxicogenomic profiling is very useful in providing an adjunct
to animal studies, there are certain precautions in interpreting microarray data
because “the transcriptome profile is extremely sensitive to any subtle changes
surrounding cells, tissues, or individual organisms” (Shoida, 2004, p. 22). There
are several such factors that may influence the outcome of a microarray experi-
ment and bias the toxicant signature. For example, circadian rhythms are often
overlooked. A simple change out of culture media can have a dramatic effect on
cells in culture.
In conclusion, gene expression array technologies have been largely accepted in
the scientific community. They are well suited as tools for drug discovery and the
elucidation of drug-target mechanism(s) of action (Cunningham, 2000; Clarke et al.,
2001). In particular, microarrays provide insight into

1. The discovery and validation of new targets

2. Determination of drug efficacy, resistance, and toxicity
3. Identification of new diagnostic biomarkers

© 2010 Taylor & Francis Group, LLC

16 Genomic and Proteonomic Microarray Technology in Drug Discovery

PROTEOMICS TODAY—THE GREAT CHALLENGE

Riding upon the success of the Human Genome Project in cataloging the 30,000 to
40,000 genes of the human genome, the Human Proteome Organization (HUPO;
http://www.hupo.org) was established to catalog the entire set of expressed proteins
comprising the human proteome(s), the Human Proteome Project (HPP). The orga-
nization has grown to address 13 separate initiatives for targeted research, for exam-
ple, Plasma Proteome Project (PPP), Human Antibody Initiative (HAI), Disease
Biomarkers Initiatives (DBI), and so forth.
As Tyers and Mann (2003, p. 193) related,

Proteomics would not be possible without the previous achievements of genomics,

which provided the “blueprint” of possible gene products that are the focal point of
proteomics studies.

For the development of new therapeutics the proteome is an important quest

given that proteins make up the majority of drug targets. Yet, the proteome is
perhaps greater than an order of magnitude more complex than the genome
(Figure 1.12). While the genome contains 10 4 to 105 genes (including splice vari-
ants), the proteome may contain over a million proteins if post-translational modi-
fications and isotypes (isoforms) are included. Furthermore, proteins vary in wide
abundance and occur in multi-protein complexes localized within cellular and sub-
organelle membranes.
The real challenge could be in the high-throughput, highly parallel, micro-
preparation of this structurally diverse class of biomolecules in their native states.
The assessment of low abundance proteins has required the adoption of enrich-
ment strategies prior to detection by mass spectrometry (Figure 1.13). In a number
of cases, protein complexes require isolation with associated membrane components
in order to preserve activity. It will also be important to be able to reassemble these
multi-protein complexes in their native and active states.
Yet, there are those who believe that because of its excellent sensitivity, mass
spectrometry (mass spec) may be able to detect proteins in biological fluids such as
serum without the need for separation. Petricoin and Liotta (2004) review the mass

Proteomics Today
The Great Challenge
“Defining the entire set of proteins of an organism”
Separate
Proteome Enrich
Genome
Classify
Sequence
Structure-Function

105–106 “proteins” 104–105 “genes”

FIGURE 1.12 Proteomics today—the great challenge.

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 17

Need: Complexity Reduction

Affinity Capture
Protein Abundance Class separation
33K < x < 106 proteins
Fractionation

“microarray”

Enrichment

Analysis
Mass Spec
M
ELISA
Phage Display

FIGURE 1.13 The need for complexity reduction.

spec differential display pattern profiling of serum proteins and peptides associated
with various cancers. Protein profiling offers an exciting opportunity as a noninva-
sive (or nearly so with a prick of blood) diagnostic tool similar in scope to that of the
magnetic resonance imaging (MRI) scan for body tissues.

THE POTENTIAL ROLE FOR PROTEIN

MICROARRAYS IN DRUG DISCOVERY
As we previously pointed out there are some very good reasons to consider pro-
teomic approaches in drug discovery:

1. Most drug targets are proteins (largely receptors and enzymes).

2. Drug-mediated therapy is based upon less than 500 targets (a very small
fraction of the proteome’s estimated +100,000 to 106 proteins); therefore,
finding many more effective targets is likely and highly desirable.
3. mRNA profiling is an indirect assessment of protein expression and cannot
detect post-translational modification, an important signaling and regula-
tory process for drug targeting.

Because a modest number of well-characterized antibodies are readily available, it

is no surprise that the first demonstrations in the use of protein microarrays have
come from work on antibody-antigen arrays. Yet, the fundamental technology is
not new. Roger Ekins (see reviews—Ekins et al., 1990; Ekins, 1998) introduced the
microspot technology in the 1980s for clinical diagnostics. Later, MacBeath and
Schriber (2000) and Haab et al. (2001) borrowing the tools and know-how from the
cDNA microarray world promoted the use of slide-based antibody microarrays.

© 2010 Taylor & Francis Group, LLC

18 Genomic and Proteonomic Microarray Technology in Drug Discovery

Antibody Antigen Receptor Chaperone Aptamer Enzyme

FIGURE 1.14 Protein microarray formats.

Huels and co-workers (2003) have identified several areas within the drug devel-
opment process where protein biochips have potential application. The antibody-
based microarray ELISA format is now commonplace. Antigen arrays have been
used to bait the capture and characterization of additional antibodies needed to fill
in the proteome libraries. Several well-characterized antibodies are usually required
in order to cover more than one epitope on each antigen. This is especially important
if the sandwich assay is to be optimally employed. Antibody and antigen microar-
ray formats are briefly described in more detail in Chapter 6, “Protein Microarray
Applications,” as well as in Chapter 7, “Multiplex Assays.”
Future generation protein arrays will include in addition to antibody-antigen
binding, other protein-protein or receptor-ligand interactions (Figure 1.14). These
include protein-peptide, substrate-enzyme, aptamer, and protein–small molecule
high-throughput assays. Proofs of principle on a variety of these formats have been
the subject of numerous reviews. Essentially, protein microarrays are believed to
be able to broadly cover the various phases of the drug discovery process simi-
larly to what we have described for gene-expression microarrays (Figure 1.15).
However, as with the DNA microarray, there remain a few hurdles ahead of us
(Figure 1.16).

Critical Issues with Protein Microarrays

Stability and Performance
While we can appreciate those first demonstrations of the utility for the protein
microarray, in retrospect, they did not perform particularly well relative to a standard
ELISA. As reported by Haab et al. (2001), only 50% of the antigens and only 20% of
the antibodies performed well (i.e., quantitative detection of the cognate antigen/anti-
body) in the µg/mL range with some allowing detection into the sub-ng/mL range.

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 19

Living Cell
Array

Target II
Discovery

I III

Toxicity
Protein Target testing
Array Validation

Gene Expression
Array

FIGURE 1.15 The role for microarrays in the drug discovery process.

Today, microarray-based immunoassays offer high sensitivity and large dynamic

ranges at levels comparable to or better than conventional “singleplex” ELISAs.
As others have pointed out, assembling proteins on a substrate is a relatively easy
task but much more of a challenge if all are to remain fully functional (Cahill, 2001;
Sreekumar & Chinnaiyan, 2002; Valle and Jendoubi, 2003). Unlike the DNA array
where the capture of cDNA or cRNA targets is relatively straightforward due to
the structural similarity between probes and targets, proteins (even within the same

Critical Issues
DNA Microarrays Protein Microarrays
Cross-platform standardization MIAME No PCR equivalent technology RCA
Non-specific adsorption
Improved printing technology 1
Cross-reactivity 2
Guidelines on statistical methods of analysis
Detection sensitivity & dynamic range RLS
Continued access to “content” Where to obtain “content” and at what cost
MIAME = Minimum Information About a Microarray Experiment
October 14, 2002 Adoption of MIAME for array standardization
Major scientific journals (including Cell, The Lancet, Nature and Science)
endorse MIAME guidelines for publishing microarray gene expression data.

RCA = rolling circle amplification; see Figure 6.18

RLS = resonance light scattering; see Figure 6.23
1 see Chapter 4. 2 see Chapter 6.

FIGURE 1.16 Critical issues facing DNA and protein microarrays.

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20 Genomic and Proteonomic Microarray Technology in Drug Discovery

class, e.g., antibodies) can vary considerably in their physical-chemical stability and
binding character on surfaces. Another challenge remains in the selection of the
optimal substrate(s), immobilization and presentation (orientation) of the ligand, as
well as the development of sensitive assays for different protein classes.

Content
Antibody coverage of the human proteome is estimated to be about 5 to 10% of
all human proteins and isoforms (Valle and Jendoubi, 2003). A major bottleneck
in the use of protein expression arrays has been the lack of such a comprehensive
set of these capture agents (Hanash, 2003). Since the equivalent of the polymerase
chain reaction (PCR) process for mass amplification of low abundant proteins does
not exist, the remaining library of proteome capture ligands may need to be gener-
ated by other means such as recombinant protein expression systems (Cahill, 2001).
However, recombinant antibodies may be less stable and have lower binding affini-
ties than monoclonal antibodies (Valle and Jendoubi, 2003). Therefore, in order to
fully implement the microarray format a host of diverse capture agents could be
required in addition to antibodies. These include peptides, small molecules, aptam-
ers, ribozymes, or other molecular recognition probes yet to be discovered. However,
it is also understandable because of the diverse nature of proteins that technologies
besides microarrays will be used in proteomics research (Hanash, 2003).

Detection
As difficult as it is going to be to isolate and produce thousands of high affinity and
specificity protein ligands, it may be even harder to come up with a good way to moni-
tor binding of proteins to the chip (Kodadek, 2001).

In the review by Kodadek (2001) the following points are made regarding near-
term approaches for detection on protein microarrays:

1. Dye labeling of proteins in cell extracts—A well-known problem is that

different proteins have different labeling efficiencies; generating individual
calibration curves for each protein within the array would be impractical, so
quantification of protein arrays will be a challenge. Note: another approach
would be to generate a labeled standard reference protein mixture or cali-
bration set at different concentrations (Haab et al., 2001). These could then
be mixed in with the cell extract providing some degree of normalization
semi-quantitative information from the dye ratios. Schroder et al. (2010)
utilized dual-color labeling in proteomic profiling experiments involving a
microarray of 810 antibodies against differentially expressed cancer protein
targets. Dual-color labeling was found to offer greater reproducibility over
that of single-color labeling.
2. Chemical modification of proteins can lead to denaturation and aggrega-
tion. This can reduce both specificity (increased nonspecific binding) and
sensitivity (decreased ligand affinity).
3. Sandwich assay—This format works well for ELISA. The success and
potential shortcomings for microarrays are discussed in the next section.

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Omics and Microarrays Revisited 21

4. Mass spectrometry—Excellent sensitivity but of low throughput and semi-

quantitative. The ICAT (isotope-coded affinity tag) labeling method for
proteins enables differential display analysis using mass spectrometry
(Griffin and Abersold, 2001). This might be adaptable to work with protein
microarrays using MALDI-TOF mass spectrometry similar to the origi-
nal method developed by Sequenom for SNP detection. Pan et al. (2009)
describe the use of internal standards of stable isotope labeled peptides to
achieve targeted protein quantification by mass spectrometry.

Micro-ELISA Formats
Assays involving antibody or antigen arrays tend to be micro-ELISA formats. For
instance, anti-cytokine monoclonal-monoclonal or monoclonal-polyclonal anti-
body pairs are readily available and well characterized for use in ELISA assays.
Cytokines are important biological indicators used in drug discovery and toxicity
testing. Therefore, it is no surprise that many of the early antibody microarray dem-
onstrations involved the determination of cytokines from cell culture. They simply
miniaturized and multiplexed the familiar sandwich immunoassay. The microarray-
based cytokine assays generally claim sensitivities in the low pg/mL range with lin-
ear dynamic ranges from about 10 to 10,000 pg/mL depending upon the analyte.
These are similar in performance to the standard ELISA.
Woodbury et al. (2002) developed a micro-ELISA assay for determination of
hepatocyte growth factor (HGF) in human serum. In this case, a horseradish peroxi-
dase (HRP)-catalyzed TSA-biotin amplification with streptavidin-Cy3 reporter was
employed. They claimed sensitivity to 0.5 pg/mL or 6 fM HGF with a linear dynamic
range of 12 to 4000 pg/mL in serum. This was good enough because clinically rel-
evant levels varied 199 to 1640 pg/mL (breast cancer patients) and 153 to 998 pg/mL
for age-matched normal controls. In addition, HGF and four other antigens found in
serum were simultaneously quantified even though the analytes varied in physiologi-
cal concentration from 20 to 60,000 pg/mL. The HGF microarray-ELISA correlated
(r 2 = 0.90) with a standard 96-well-plate ELISA.
So, for the determination of a limited set of analytes, the antibody microarray
works reasonably well and is comparable in performance if not better than that
of a standard ELISA for individual analytes. Furthermore, we will discover that
a suitable number of detection technologies are available for use on a variety of
supports. The real problem may be in at what point antibody microarrays “hit the
wall” for multiplexing. What is not generally appreciated is that each antibody
pair needs to be matched up and cross-reactivity for all pairs determined. That
involves not only the cross-reactivity of each capture antibody but also the cross-
reactivity of all secondary antibodies, including the extent of secondary to second-
ary interactions. It is not uncommon to sort through several antibody pairs before
finding compatible sets. Consider now having to do the same for an antibody array
including hundreds to thousands of analyte-specific pairs. See Chapter 6 (“Protein
Microarray Applications”) for additional examples and discussion concerning the
issue of cross-reactivity.

© 2010 Taylor & Francis Group, LLC

22 Genomic and Proteonomic Microarray Technology in Drug Discovery

Protein Profiling Formats

Protein expression profiling (protein differential display) using microarrays is con-
sidered by most to be an important tool for proteomic discovery. It is similar in
concept and approach to that of the gene expression microarray for mRNA profiling.
Sreekumar and Chinnaiyan (2002) describe a general approach to using the micro-
array in order to monitor protein expression in cancer and normal tissues. Here are
the steps:

1. Extract total protein from cancer and normal cells using a detergent (e.g.,
1% NP40).
2. Remove excess detergent from lysate by adsorption onto a solid phase
(e.g., beads).
3. Determine total protein concentration for each lysate.
4. Label equal amounts of protein from cancer and normal cell lysate (e.g.,
Cy5-NHS labeling of cancer lysate; Cy3-NHS labeling of normal lysate).
5. Remove free dye from dye-labeled proteins by gel filtration chromatography.
6. Mix Cy5-protein with Cy3-protein purified lysates.
7. Concentrate mixture.
8. Apply concentrate to antibody array.
9. Wash array free of unbound antigens, and then perform confocal scan.
10. Analyze data.

Here are some potential issues to consider: First, detergent extraction can be
problematic. Not all proteins will extract or if so to the same extent. The amount
of protein present can influence the efficiency and stability of detergent micelle
formation. Inefficient removal of detergent as well as irreversible partitioning of
proteins onto a solid support during purification is likely. If there were differ-
ences in protein abundance between test and control cell lysates before process-
ing, there could be significant differential loss following processing. Individual
proteins can have very different labeling efficiencies depending upon protein con-
centration, pH, ionic strength, and the number and accessibility of dye-reactive
amino acid residues (Kodadek, 2001). As with the labeling of nucleic acids, Cy5
and Cy3 or other dyes may have different labeling efficiencies for the same pro-
tein. Dye-labeled proteins may differentially adsorb onto the solid phase used for
purification. Concentrating may do more harm than good if proteins denature and
aggregate forming protein complexes. If such complexes are applied to the anti-
body array both false positive and false negative associations are likely.

Near-Term Biomedical Applications

Cytokines
An abundance of vendors offer antibody-based microarrays for multiplexed cyto-
kines analysis (see Chapter 2, “Commercial Microarrays,” for commercial sources).
Cytokines are biomarkers involved in cancer, cell injury, inflammation, and apop-
tosis. They are released by cells in culture in response to drug action (e.g., Turtinen

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 23

et al., 2004) or are elevated in serum in various disease states. Moreover, there are
numerous cytokines involved in cellular response with many serving as dual effec-
tors (Asao and Fu, 2000). As a result, anti-cytokine microarrays are being evalu-
ated in drug discovery for off-target toxicity testing to replace standard ELISA plate
formats. An extensive listing of cytokines is available at COPE (Cytokines & Cells
Online Pathfinder Encyclopedia) at http://www.copewithcytokines.org. Version 29.0
containing over 29,000 entries was released in spring/summer 2012.
Huang et al. (2002) prepared an antibody array for the simultaneous detection of
43 cytokines. They were able to verify the down-regulation of MCP-1 cytokine in
transfected cells (human glioblastoma cells transfected with cx43 expression vec-
tor) relative to control cells. The antibody array is an emerging technology. At least
in one study based upon the use of a commercial membrane format, the cytokine
microarray failed to accurately determine cytokine levels in bacterial and LPS-
stimulated whole human blood (Copland, 2004).

Autoimmune Diseases and Allergy

Advancement in autoimmune and inflammatory disease treatment and diagnosis
represents a critical worldwide need ranking only behind cardiovascular disease and
cancer in importance to the medical practitioner. The list of related diseases is long
but major categories include rheumatoid arthritis, asthma, diabetes type I, multiple
sclerosis, and inflammatory bowel disease, such as celiac disease.
Antigen arrays also described as “reverse-phase protein array” (Paweletz et
al., 2001) involve the immobilization of proteins to serve as bait for various protein-
protein interactions (Sreekumar et al., 2002). For example, Joos et al. (2000) printed
various auto-antigens present in sera with known association with various autoim-
mune diseases (e.g., Grave’s disease, lupus, connective tissue disease, and others).
The group then screened various sera for the presence of the auto-antibodies. By
immobilizing on the array a serial dilution series for each antigen, the titers for these
antibodies could be determined.
Feng et al. (2004) prepared an antigen microarray on polystyrene support
including 15 auto-antigens useful for the detection of auto-antibodies involved in
rheumatoid autoimmune diseases. In other studies, de Vegvar et al. (2003) used
antigen microarrays to examine epitope-specific antiviral antibody responses in
vaccine trials in an animal model for human immunodeficiency virus (HIV) infec-
tion. Huber et al. (2002) has reviewed different formats for antigen microarrays.
The review by Robinson (2006) examines autoimmune disease, cancer, infec-
tious diseases, and allergen profiling with antigen microarrays. Mezzasoma and
co-workers (2002) used antigen microarrays to determine the levels of infectious
agents in human sera. Antigens (Toxoplasma gondii, rubella virus, cytomegalo-
virus, herpes simplex virus, ToRCH antigens) were printed in an array format.
Serum samples were applied and serodiagnosis determined using a sandwich assay
employing fluorescently labeled secondary antibodies directed toward the primary
sera antibodies. The Utz Lab has additional information regarding antigen arrays
on their Stanford School of Medicine Web site (http://www.stanford.edu/group/
utzlab/autoantibodies.htm).

© 2010 Taylor & Francis Group, LLC

24 Genomic and Proteonomic Microarray Technology in Drug Discovery

The typing of various allergens using the antigen microarray has also met with
success due in part to the availability of recombinant allergens (i.e., content). Jahn-
Schmid and co-workers (2003) examined the analytical performance of an allergen
(grass and tree pollens) microarray for the detection of allergen-specific serum IgG
in 51 patient sera. While considerable variation was observed with intra- and inter-
assay for some allergens, the sensitivity and specificity of the microarray were com-
parable to the conventional ELISA. Shreffler et al. (2004) constructed an antigen
array including an overlapping series of peptide probes representing epitopes asso-
ciated with the major peanut allergens. Examining 77 patient sera the group found
considerable variation in patient IgE–epitope profiling suggesting such population
heterogeneity might be of prognostic value.
Finally, Nishizuka et al. (2003) undertook the arduous task of proteomic profil-
ing the NCI-60 cancer cell lines based upon high-density arraying of cell lysates.
In this case, lysates are prepared in a urea denaturing buffer and maintained in
a reduced state with dithiothretol (DTT). This allows opportunity for additional
assessment from 2D-PAGE gels. Serial dilutions of each protein lysate were printed
onto the substrate (FAST slides, nitrocellulose-coated glass, Schleicher & Schuell,
Germany). Monoclonal antibodies screened by Western blotting to lysate were
used for detection and SYPRO ruby protein stain (molecular probes) for determi-
nation of total protein. Fifty-two proteins (i.e., those with high specificity antibody
recognition) were analyzed in lysates. Of these, 31 were matched to cDNA and
GeneChip microarrays; 19 of those 31 showed significant correlation between the
two gene-expression formats used earlier in characterizing the NCI-60 cell lines
(e.g., Ross et al., 2000; Scherf et al., 2000). How do the expressions of these pro-
teins correlate with the corresponding mRNA profiles? And the answer, please...?
Structure-related proteins are almost always better correlated with mRNA levels
across the 60 cell lines.

FUTURE MEDICINE—PHARMACOPROTEOMICS
OR PHARMACOGENOMICS?
The end result for drug development is to successfully supply cost-effective drugs
that provide for better patient treatment, offering cures from new therapies and
improvements in disease management. The latter includes the advancement of diag-
nostics with tests that will more rapidly and more accurately determine disease state
and monitor treatments. But, what road should we travel down in the future—phar-
macogenomics or pharmacoproteomics (Figure 1.17)?
Jain (2004) defines pharmacoproteomics as the use of proteomic technologies
in drug discovery and development. It is Jain’s contention that pharmacoproteomics
rather than genotyping will take the lead role in promoting the practice of personal-
ized medicine. This remains to be seen. Key to that success will be the continued
application of protein chips, enabling future discovery and development of drugs for
personalized therapy, and entry into point-of-care diagnostics.
However, do not rule out pharmacogenomic approaches (Ginsburg et al.,
2001). Sabatini’s reverse-transfection method of creating “live cell” microarrays

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 25

Genome Proteome

Gene i.d. Protein i.d.

Gene functions Protein functions

Targets
Polymorphisms
Post-translational
modifications
Pathways

Pharmacogenomics Pharmacoproteomics

Molecular Medicine

FIGURE 1.17 Where are we going from here?

offers even greater advantage for drug discovery and development (Ziauddin and
Sabatini, 2001). The method, relying on arrayed cDNA expression vectors to trans-
form adherent cells, provides localized, real-time gene expression analysis of the
putative gene product. The live cell microarray could eventually displace the use
of protein expression microarray for identifying drug targets. This is provided
that more extensive libraries of full length cDNAs (needed to express the com-
plete protein) become available. The approach could potentially be utilized for
high-throughput screens to uncover genotype-phenotype relationships. Offering
an alternative cellular approach, in 2011, the NIH and DARPA announced pro-
grams with the collaboration of the FDA to produce “body-on-a-chip” platforms
of living cell types arrayed on microfluidic devices for detecting the toxicity of
candidate drugs.
What appears to be certain is that microarrays in one omics-based form or another
(DNA, protein, cell, tissue, or small molecule) are playing an increasingly important
role in drug development and diagnostics (Figure 1.18). Perhaps omics will eventu-
ally evolve into an integrated “systems biology” approach (Ideker et al., 2001), one
in which we monitor metabolic pathways, transport, compartmentalization, degrada-
tion, and so forth, and their inter-relationships for small molecules, cell surfaces (e.g.,
carbohydrate microarrays) (Wang et al., 2002), and biopolymers alike (Figure 1.19).
We are just at the beginning of molecular medicine (personalized medicine) and
molecular diagnostics (companion diagnostics). It remains an exciting era for sci-
ence, technology, and personalized medicine.

© 2010 Taylor & Francis Group, LLC

26 Genomic and Proteonomic Microarray Technology in Drug Discovery

Gene Expression Gene Discovery

p53
Profiling Pathways
Ion channels
Toxicity (off-target)
P450; NO
SNP/Polymorphism Forensics/paternity/military
RNA/DNA Screening i.d.
Pharmacogenomics HLA
Predictive CF
Genotyping Population Screening TB
Infectious Disease BRACA
tumor
Genetic Disease Disease-state monitoring metastasis
Cancer Alzeheimer’s

Protein Expression Pathways

Profiling Drug Discovery
Toxicity (off-target)
Microarray Proteins Cytokines
(IL, TNF)
Technologies Diagnostic Immunoassay Infectious Disease Apoptosis
Cellular proteins (caspases
Secreted proteins kinases
phosphatases)
Receptor-Ligand Drug Discovery
Binding Assay

Phenotyping Pharmacogenomics

Ovarian cancer
Ion channels
Cells/ Diabetes
Tissue Inflammatory
Inter-cellular Toxicity (off-target) Diseases
signaling pathway
Prognostic Cancer new drugs

Receptor-Ligand
Binding Assay Drug Discovery

Technology Application Examples

FIGURE 1.18 Microarray applications.

DNA Arrays Cell/Tissue Arrays Protein Arrays

Systems Biology

Carbohydrate
Patient
lipids
History
Small molecules
Ions

Molecular Diagnostics

The future of the biochip rests within its ability to serve

as a fully integrated device for multiplexed analysis

FIGURE 1.19 Molecular diagnostics.

© 2010 Taylor & Francis Group, LLC

Omics and Microarrays Revisited 27

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Other documents randomly have
different content
Rousseau, though I admire his style and genius; yet if humanity
were indebted to him for no more than this sagacious counsel, and
the felicity conferred by his maxims on early childhood, it would still
suffice to make us love and consider him as a benefactor.
But our new acquisition of a handsome staircase did not
exclusively occupy the whole of our time; as in our solitude we had
nothing to consider but our own pleasure or convenience, and our
daily wants were not subject to the occurrence of other social duties,
we saw no occasion for tormenting ourselves with a greater degree
of labour in every day than was wholesome for our bodily health. We
had no harsh surveyor, no inquisitive examiners; no troublesome
neighbours nor counsellors. If we occasionally regretted not being
members of a large society under just laws and agreements
established between societies of men, we more frequently
complimented ourselves on not being subjected to this restraint and
the inconveniences that arise from it. If we happened now and then
to feel the want of some of the high-wrought pleasures of social
existence, we were speedily solaced by reflecting that we did not
stand in need of money; that we had no uneasy care about the
acquisition of it; that we excited neither envy, pity, nor censure;
while the imperfection of our achievements, and the trouble they
cost us, were richly compensated by the freedom and cheerfulness
with which they were executed, ever without altercation, and with
united hearts and souls.
I will briefly narrate the few remarkable occurrences that took
place during the construction of our staircase.
A few days after the commencement of our stair-case, the two
she-goats gave us two kids, and our ewes five lambs; so that we
now saw ourselves in possession of a pretty flock: but lest the
domestic animals should follow the example of the ass, and run
away from us, I tied a bell to the neck of each. We had found a
sufficient number of bells in the vessel, which had been shipped for
trading with the savages; it being one of the articles they most
value. We could now immediately trace a deserter by the sound, and
bring it back to the fold.
 Next to the winding stairs, my chief occupation was the
management of the young buffalo, whose wound in the nose was
quite healed, so that I could lead it at will with a cord or stick passed
through the orifice, as the Caffrarians do. I preferred the latter,
which answered the purpose of a bit; and I resolved to break-in this
spirited beast for riding as well as drawing. It was already used to
the shafts, and very tractable in them; but I had more trouble in
inuring him to the rider, and to wear a girth, having made one out of
the old buffalo’s hide. I formed a sort of saddle with sail-cloth, and
tacked it to the girth. Upon this I fixed a burthen, which I increased
progressively. I was indefatigable in the training of the animal, and
soon brought it to carry, without fear or repugnance, large bags full
of potatoes, salt, and other articles, such as the ass had patiently
borne to be loaded with. The monkey was his first rider, who stuck
so close to the saddle, that in spite of the plunging and kicking of
the buffalo, it was not thrown. Francis was then tried, as the lightest
of the family; but throughout his excursion I led the beast with a
halter, that it might not throw the child off. Jack now showed some
impatience to mount the animal in his turn. Some restraint was
requisite:—I passed the appropriate piece of wood through the
buffalo’s nose, and tied strong packthread at each end of the stick,
bringing them together over the neck of the animal; and I then put
this new-fashioned bridle into the hands of the young rider, directing
him how to use it. For a time the lad kept his saddle,
notwithstanding the repeated jumps of the horned steed; at length a
side jolt threw him on the sand, without his receiving much injury.
Ernest, Fritz, and lastly myself, got on successively, with more or less
effect. His trotting shook us to the very centre, the rapidity of his
gallop turned us giddy, and our lessons in horsemanship were
reiterated many days before the animal was tamed, and could be
rode with either safety or pleasure. At last, however, we succeeded
without any serious accident; and the strength and swiftness of our
saddled buffalo were prodigious. It seemed to sport with the
heaviest loads. My three eldest boys mounted it together now and
then, and it ran with them with the swiftness of lightning. By
continued attentions it at length became extremely docile: it was not
in the least apt to start; and I really felt satisfaction in being thus
enabled to make my sons expert riders, so that if they should ever
have horses, they might get on the most restive and fiery without
any fear:—none could be compared to our young buffalo; and the
ass which I had intended to employ in the same way was far
surpassed by this new member of our family. Fritz and Jack, with my
instructions, amused themselves in training the animal as horses are
exercised in a riding-house; and by means of the little stick through
the nose, they were able to do what they pleased with him.
In the midst of all this Fritz did not neglect his eagle; he daily shot
some small birds which he gave it to eat, placing them sometimes
betwixt the buffalo’s horns, sometimes on the back of one of the
hens, or of a flamingo, or on a shelf, or at the end of a stick, in order
to teach it to pounce like a falcon upon other birds. He taught it to
perch on his wrist whenever he called or whistled to it; but some
time elapsed before he could trust it to soar without securing its
return by a long string, apprehending its bold and wild nature would
prompt it to take a distant and farewell flight from us.
Our whole company, including even the inert Ernest, was infected
with the passion of becoming instructors. Ernest tried his talents in
this way with his monkey; who, it must be confessed, seldom failed
to furnish him with work. It was no poor specimen of the ludicrous
to see the lad; he whose movements were habitually slow and
studied, now constrained to skip, and jump, and play a thousand
antics with his pupil during training hours, and all the time deeply
interested in carrying forward the lesson the grotesque mimic was
condemned to learn, of carrying small loads, climbing the cocoa-
trees, and to fetch and bring the nuts. He and Jack made a little
hamper of rushes, very light: they put three straps to it, two of
which passed under the fore, and one between the hind legs of the
animal, and were then fastened to a belt in front, to keep the
hamper steady on the back of the mischievous urchin. This
apparatus was at first intolerable to poor Knips: he gnashed his
teeth, rolled on the ground, jumping like a mad creature, and did
every thing to get rid of it; but all in vain, for education was the
standing order, and he soon found he must submit. The hamper was
left on day and night; its sole food was what was thrown into it; and
in a short time pug was so much accustomed to the burden, that he
began to spit and growl whenever we attempted to take it off, and
every thing given to the creature to hold was instantly thrown into it.
Knips became at length a useful member of our society; but he
would only obey Ernest, whom he at once loved and feared, thus
affording a proof of at least one of the great ends of all instruction.
Jack was less successful with his little jackal, which he had named
Hunter, hoping that its qualities would justify the name. He made
continual attempts to induce the animal to go after game; but for
the first six months he advanced no further in the lesson than
teaching him to bring what was thrown to it: and when it was dead
game, Hunter was sure to devour it on the way, and to bring home
the skin alone: but it was nevertheless so pretty and tractable a
creature, that I intreated the boy not to relinquish a task that would
prove so beneficial to us; and he persevered with considerable zeal.
These different occupations filled up several hours of the day;
when, after working at our stairs, we assembled in the evening
round our never-failing constant friend, the good mother, to rest
ourselves: and forming a little circle, every individual of which was
affectionate and cheerful, it was her turn to give us some agreeable
and less fatiguing occupation in the domestic concerns of Falcon
Stream: such, for example, as endeavouring to improve our candle-
manufactory, by blending the berry and the bees-wax, and
employing the reed-moulds invented by Jack: but having found
some difficulty in taking out the candles when cold, I adopted the
plan of dividing the moulds, cleaning the inside, and rubbing it over
with a little butter, to prevent the wax from adhering to it; then to
rejoin both halves with a band that could be loosened at pleasure, to
facilitate the extraction of the tapers. The wicks gave us most
trouble, as we had no cotton. We tried with moderate success the
fibrous threads of the karatta, and those of the algava or flame-
wood; but each had the inconvenience of becoming a sort of coal or
cinder. The production which gave us the most satisfaction was the
pith of a species of elder; but it did not, however, lessen our desire
to discover the only appropriate ingredient, the cotton-tree. I
likewise contrived a method of rendering our candles even and
shining, by rolling them between two boards; they now were only
distinguishable from those of Europe by a greenish hue. On my
observing to my sons that wax was bleached like linen, by spreading
it on cloths, and exposing it to the dew and sun, they wished to try
the process; but as our green tapers burned remarkably well,
bleaching the wax would have been a useless luxury and loss of
time, which I could turn to more account in manufacturing our
impenetrable boots without seams, of the caoutchouc or elastic
gum.
I began with a pair for myself; and I encouraged my children to
afford a specimen of their industry, by trying to form some flasks
and cups that could not break. They commenced by making some
clay moulds, which they covered with layers of gum, agreeably to
the instructions I had given them.
In the meanwhile I compactly filled a pair of stockings with sand,
and covered them with a layer of clay, which I first dried in the
shade, and afterwards in the sun. I then took a sole of buffalo-
leather, well beaten, and studded round with tacks, which served me
to fix it under the foot of the stocking; and after this I poured the
liquid gum into all the interstices, which on drying produced a close
adhesion between the leather and stocking sole. I next proceeded to
smear the whole with a coat of resin of a tolerable thickness; and as
soon as this layer was dried on, I put on another, and so on till I had
spread on a sufficiency with my brush. After which I emptied the
sand, drew out the stocking, removed the hardened clay, shook off
the dust, and thus obtained a pair of seamless boots, as finished as
if made by the best English workman; being pliant, warm, soft,
smooth, and completely water-proof.
I hung them up directly, that they might dry without shrinking.
They fitted uncommonly well; and my four lads were so highly
pleased with their appearance, that they skipped about with joy in
requesting me to make each of them a pair. I refrained from any
promise, because I wished to ascertain their strength previously, and
to compare them with boots made out of mere buffalo-leather. Of
these I at once began a pair for Fritz, with a piece of the slaughtered
buffalo’s skin. They gave far more trouble than those manufactured
with the caoutchouc, which I used to cover the seams and render
them less pervious to water. The work turned out very imperfect,
and so inferior to my incomparable boots, that Fritz wore them
reluctantly; and the more so, as his brothers shouted with laughter
at the difficulty he had to run in them. My boys had succeeded
tolerably well with their new ware, though still imperfect; but as a
first essay performed by tyro artists, I was satisfied with their
productions.
We had also been engaged in the construction of our fountain,
which afforded a perpetual source of pleasure to my wife, and
indeed to all of us. In the upper part of the stream we built with
stakes and stones a kind of dam, that raised the water sufficiently to
convey it into the palm-tree troughs; and afterwards, by means of a
gentle slope, to glide on contiguous to our habitation, where it fell
into the tortoise-shell bason, which we had elevated on stones to a
certain height for our convenience; and it was so contrived that the
redundant water passed off through a cane pipe fitted to it. I placed
two sticks athwart each other for the gourds, that served as pails, to
rest on; and we thus produced, close to our abode, an agreeable
fountain, delighting with its rill, and supplying us with a pure crystal
fluid, and such as we frequently could not get when we drew our
water from the bed of the river, which was often blended with the
leaves and earth fallen into it, or rendered turbid by our water-fowls.
The only inconvenience was, that the water flowing in this open
state through narrow channels in a slender stream, was heated, and
not refreshing when it reached us. I resolved to obviate this
inconvenience at my future leisure, by employing, instead of the
uncovered conduits, large bamboo-canes fixed deep enough in the
ground to keep the water cool. In waiting the execution of this
design, we felt pleasure in the new acquisition; and Fritz, who had
suggested the notion, received his tribute of praise from all.
 CHAPTER XXX.
The wild ass;—difficulty in breaking it;—the heath-
fowl’s nest.

We were scarcely up one morning, and had got to work in putting

the last hand to our winding stair-case, when we heard at a distance
two strange peculiar kind of voices, that resembled the howlings of
wild beasts, mixed with hissings and sounds of some creature at its
last gasp, which I was at a loss to explain, and I was not without
uneasiness; our dogs too pricked up their ears, and seemed to whet
their teeth for a sanguinary combat with a dangerous enemy.
From their looks we judged it prudent to put ourselves in a state
of defence; we loaded our guns and pistols, placed them together
within our castle in the tree, and prepared to repel vigourously any
hostile attack from that quarter. The howlings having ceased an
instant, I descended from our citadel, well armed, and put on our
two faithful guardians their spiked collars and side-guards: I
assembled our cattle about the tree to have them in sight, and I
reascended to look around for the enemy’s approach. Jack wished
they might be lions—I should like, said he, to have a near view of
the king of beasts, and should not be in the least afraid of him, for
lions are deemed generous!
I do not advise you, answered I, to trust the report, though you
may not fear a lion when elevated as you are forty feet above them:
but these are not lions; their roarings are more lengthened,
majestic, and fill all other animals that hear them with fear and
trembling; I do not observe this effect amongst ours.
 Fritz.—I rather surmise they are a troop of jackals, disposed to
avenge the death of their comrades.
Ernest.—It is not the jackal’s cry: I am more inclined to fear they
are hyenas, whose howling must, one would think, be as frightful as
their looks.
Francis.—Now I think they are savages come to eat their prisoners
on our island; I wish we could save them, and get a good Man
Friday as Robinson Crusoe did.
Whatever it is, children, let us not yield to fear or imagination; we
are in safety here...
At this very instant the howlings were renewed and quite close to
us. Fritz got as near the spot as he could, listened attentively and
with eager looks, then threw down his gun and burst into a loud
laughter, exclaiming: Father, it is our ass—the deserter comes back
to us, chanting the hymn of return: listen! do you not hear his
melodious brayings in all the varieties of the gamut?—We lent an
ear; our doubts ceased, and we felt somewhat mortified at our
premature alarms and preparations of defence against such an
ignoble foe.
I on my part, however, was soon reconciled to the offence against
our pride, since it also insured our safety: and a fresh roar, in sounds
unquestionable, raised loud peals of laughter among us; and then
followed the usual train of jests and mutual banter at the alarm we
had one and all betrayed. Shortly after, we had the satisfaction of
seeing among the trees our old friend Grizzle, moving towards us
leisurely, and stopping now and then to browse; but to our great joy
we perceived in his train one of the same species of very superior
beauty, and when it was nearer I knew it to be a fine onagra18 or
wild ass, which I conceived a strong desire to possess, though at the
same time aware of the extreme difficulty there would be in taming
and rendering him subject to the use of man. Some writers who
have described it under the name of the Œigitai, (or long-eared
horse,) given it by the Tartars, affirm that the taming it has been
ever found absolutely impracticable; but my mind furnished an idea
on the subject which I was resolved to act on, if I got possession of
the handsome creature. Without delay I descended the ladder with
Fritz, desiring his brothers to keep still; and I consulted my privy-
counsellor on the means of surprising and taking the stranger
captive. I got ready, as soon as possible, a long cord with a running
knot, one end of which I tied fast to the root of a tree; the noose
was kept open with a little stick slightly fixed in the opening so as to
fall of itself on the cord being thrown round the neck of the animal,
whose efforts to escape would draw the knot closer. I also prepared
a piece of bamboo about two feet long, which I split at the bottom,
and tied fast at top, to serve as nippers. Fritz attentively examined
my contrivance, without seeing the use of it. Prompted by the
impatience of youth, he took the ball-sling and proposed aiming at
the wild ass with it, which he said was the shortest way of
proceeding. I declined adopting this Patagonian method, fearing the
attempt might fail, and the beautiful creature avail itself of its natural
velocity to evade us beyond recovery: I therefore told him my
project of catching it in the noose, which I gave him to manage, as
being nimbler and more expert than myself. The two asses drew
nearer and nearer to us. Fritz, holding in his hand the open noose,
moved softly on from behind the tree where we were concealed, and
advanced as far as the length of the rope allowed him: the onagra
was extremely startled on perceiving a human figure; it sprung some
paces backward, then stopped as if to examine the unknown form;
but as Fritz now remained quite still, the animal resumed its
composure and continued to browse. Soon after he approached the
old ass, hoping that the confidence that would be shown by it, would
raise a similar feeling in the stranger: he held out a handful of oats
mixed with salt; our ass instantly ran up to take its favourite food,
and greedily devoured it; this was quickly perceived by the other. It
drew near, raised its head, breathed strongly and came up so close,
that Fritz, seizing the opportunity, succeeded in throwing the rope
round its neck; but the motion and stroke so affrighted the beast
that it instantly sprang off; it was soon checked by the cord, which
in compressing the neck almost stopped its breath: it could go no
further, and after many exhausting efforts, it sunk panting for breath
upon the ground. I hastened to loosen the cord and prevent its
being strangled. I then quickly threw our ass’s halter over its head; I
fixed the nose in my split cane, which I secured at the bottom with
packthread. Thus I succeeded in subduing the first alarm of this wild
animal, as farriers shoe a horse for the first time. I wholly removed
the noose that seemed to bring the creature into a dangerous
situation; I fastened the halter with two long ropes to two roots near
us, on the right and left, and let the animal recover itself, noticing its
actions, and devising the best way to tame it in the completest
manner.
The rest of my family had by this time come down from the tree
and beheld the fine creature with admiration, its graceful shape and
well-turned limbs, which placed it so much above the ass, and nearly
raised it to the noble structure of the horse! In a few moments the
onagra got up again, struck furiously with its foot, and seemed
resolved to free itself from all bonds: but the pain of its nose, which
was grasped and violently squeezed in the bamboo, forced it to lie
down again. My eldest son and I now gently undid the cords, and
half led, half dragged it between two roots closely connected, to
which we fastened it afresh so as to give the least scope for motion,
and thus, render its escape impracticable, whilst it enabled us to
approach securely and examine the valuable capture we had made.
We also guarded against master Grizzle playing truant again, and
tied him fast with a new halter, confining its fore legs with a rope. I
then fastened it and the wild ass side by side, and put before both
plenty of good provender to solace their impatience of captivity.
We had now the additional occupation of training the onagra for
our service or our pleasure as might turn out to be most practicable:
my boys exulted in the idea of riding it, and we repeatedly
congratulated each other on the good fortune which had thus
resulted from the flight of our ass. Yet I did not conceal that we
should have many difficulties to encounter in taming it, though it
seemed very young and not even to have reached its full growth.
But I was inclined to think proper means had not been hitherto
adopted, and that the hunters, almost as savage as the animals
themselves, had not employed sufficient art and patience, being
probably unconscious of the advantages of either. I therefore
determined to resort to all possible measures: I let the nippers
remain on its nose, which appeared to distress him greatly, though
we could plainly perceive their good effect in subduing the creature,
for without them no one could have ventured to approach him; I
took them off however at times when I gave it food, to render eating
easier, and I began, as with the buffalo, by placing a bundle of sail-
cloth on its back to inure it to carry. When accustomed to the load, I
strove to render the beast still by degrees more docile, by hunger
and thirst; and I observed with pleasure that when it had fasted a
little and I supplied it with food, its look and actions were less wild. I
also compelled the animal to keep erect on its four legs, by drawing
the cords closer that fastened it to the roots, in order to subdue
gradually by fatigue its natural ferocity. The children came in turns to
play with it and scratch its ears gently, which were remarkably
tender; and it was on these I resolved to make my last trial if all
other endeavours failed. For a long time we despaired of success;
the onagra made furious starts and leaps when any of us went near
it, kicked with its hind feet, and even attempted to bite those who
touched it. This obliged me to have recourse to a muzzle, which I
managed with rushes, and put on when it was not feeding. To avoid
being struck by its hind feet, I partially confined them by fastening
them to the fore feet with cords, which however I left moderately
loose, that we might not encroach too much upon the motion
necessary for its health. It was at length familiarized to this
discipline, and was no longer in a rage when we approached, but
grew less impatient daily, and bore to be handled and stroked.
At last we ventured to free it by degrees from its restraints and to
ride it as we had done with the buffalo, still keeping the fore feet
tied; but notwithstanding this precaution and every preceding
means, it proved as fierce and unruly as ever for the moment. The
monkey, who was first put on its back, held on pretty well by
clinging to its mane, from which it was suspended as often as the
onagra furiously reared and plunged; it was therefore for the present
impracticable for either of my sons to get upon it. The perverse
beast baffled all our efforts, and the perilous task of breaking it was
still to be persevered in with terror and apprehension. In the stable
it seemed tolerably quiet and gentle; but the moment it was in any
degree unshackled, it became wholly ferocious and unmanageable.
I was at length reduced to my last expedient, but not without
much regret, as I resolved, if it did not answer, to restore the animal
to full liberty. I tried to mount the onagra, and just as in the act of
rearing up violently to prevent me, I seized with my teeth one of the
long ears of the enraged creature, and bit it till it bled; instantly it
stood almost erect on its hind feet, motionless, and as stiff as a
stake; it soon lowered itself by degrees, while I still held its ear
between my teeth. Fritz seized the moment and sprung on its back;
Jack, with the help of his mother, did the same, holding by his
brother, who, on his part, clung to the girth. When both assured me
they were firmly seated, I let go the ear: the onagra made a few
springs less violent than the former, and checked by the cords on its
feet, it gradually submitted, began to trot up and down more quietly,
and ultimately grew so tractable that riding it became one of our
chief pleasures. My lads were soon expert horsemen; and their
horse, though rather long-eared, was very handsome and well
broken in. Thus patience on our parts conquered a serious difficulty,
and gained for us a proud advantage.
In the name of goodness, said my wife to me one evening, after
one of our first essays, where did you learn this strange notion of
biting the animal’s ear? I learned it, replied I, from a horse-breaker
whom I fell in with by chance: he had lived long in America and
carried on the skin-trade with the savages, to whom he took in
exchange various European goods. He employed in these journeys,
half-tamed horses of the southern provinces of that country, which
are caught in snares or with nooses. They are at first unruly and
resist burthens, but as soon as the hunter bites one of their ears
they become mild and submissive; and they become so docile, that
any thing may be done with them. The journey is continued through
forests and over heaths to the dwellings of the savages; skins are
given in barter for the goods brought them, with which the horses
are re-loaded. They set out again on their return, and are directed
by the compass and stars to the European settlements, where they
profitably dispose of their skins and horses.—Till now I thought this
singular mode of taming a wild beast fabulous, but the young
onagra convinces me of the truth of the accounts I heard. In a few
weeks the onagra was so effectually tamed, that we all could mount
it without fear: I still however kept his two fore legs confined
together with the cord, to moderate the extreme swiftness of its
running. In the room of a bit, I contrived a curb, and with this and a
good bite applied, as wanted, to the ear, it went to right or left at
the will of the rider. Now and then I mounted it myself, and not
without an emotion of pride at my success in subduing an animal
that had been considered by travellers and naturalists as absolutely
beyond the power of man to tame. But how superior was my
gratification in seeing Fritz spring at any time on the creature’s back
and do what he pleased with it, drive along our avenue like
lightning, in depicting to my fond imagination that even on a desert
unknown island, I could qualify my dear children to re-enter society
and become in such respects its ornament! in beholding their
physical strength and native graces unfold themselves, and these
keeping pace with the improvement of their intelligence and their
judgement; and in anticipating that, buried as they were in a distant
retreat, far from the tumult of the world, and all that excites the
passions, their sentiments would be formed in exact conformity to
the paternal feelings of my heart! I had not lost hope that we should
one day return to Europe in some vessel chance might throw on our
coast, or even with the aid of our pinnace; but I felt at the same
time, and my wife still more, that we should not leave the island
without a lively regret, and I determined to pursue my arrangements
as if we were to close existence on a spot where all around us
prospered.
During the training of our horse, which we named Light-foot, a
triple brood of our hens had given us a crowd of little feathered
beings; forty of these, at least, were chirping and hopping about us,
to the great satisfaction of my wife, whose zealous care of them
sometimes made me smile. Most women’s hearts are so imbued with
maternal love as to excite in them a fondness for whatever bears a
similitude to infancy. Thus, my admirable partner, far from
complaining of the trouble such a number of young chickens gave
her, took delight in it, and was constantly admiring them; yet her
care and admiration did not prevent her appropriating a part of them
to the table, and sending the remainder in small colonies to feed and
breed in the desert, where we could find them as they were wanted
for our use.
Here, she said, are animals of real utility in a family, far beyond
your monkeys, jackals, and eagles, that do nothing but eat, and are
unfit to be eaten. The buffalo was not found fault with, because it
brought her the provisions, nor the onagra, on which she liked to
see her sons gallop. From the time we had trained it to this, the
rough-paced buffalo that shook us to pieces was no longer used for
riding, but kept entirely for drawing.
This increase of our poultry reminded us of the necessity of an
undertaking we had long thought of, and was not in prudence to be
deferred any longer; this was the building between the roots of our
great tree, covered sheds for all our bipeds and quadrupeds. The
rainy season, which is the winter of these countries, was drawing
near, and to avoid losing most of our stock it was requisite to shelter
it.
We began by forming a kind of roof above the arched roots of our
tree, and employed bamboo canes for the purpose; the longest and
strongest supported the roofing in the place of columns, the smaller
more closely united and composed the roof itself. I filled up the
interstices with moss and clay, and I spread over the whole a thick
coat of tar. By these means I formed a compact and solid covering,
capable of bearing pressure. I then made a railing round it, which
gave the appearance of a pretty balcony, under which, between the
roots, were various stalls sheltered from rain and sun, that could be
easily shut and separated from each other by means of planks nailed
upon the roots; part of them were calculated to serve as a stable
and yard, part as an eating-room, a store-room, &c., and as a hay-
loft to keep our hay and provisions dry in.
This work was soon completed; but afterwards it was necessary to
fill these places with stores of every kind for our supply throughout
the wet season. In this task we engaged diligently, and went daily
here and there with our cart to collect every thing useful, and that
might give us employment whilst the weather confined us to the
house.
One evening on our return from digging up potatoes, as our cart
loaded with bags, drawn by the buffalo, ass and cow, was gently
rolling along, seeing still a vacant place in the vehicle, I advised my
wife to go home with the two youngest boys whilst I went round by
the wood of oaks with Ernest and Fritz to gather as many sweet
acorns as we could find room for. We had still some empty sacks.
Ernest was accompanied by his monkey, who seldom left him; and
Fritz, horseman like, was on his dear onagra, which he had
appropriated to himself, inasmuch as he had helped to take and
tame it, and indeed because he knew how to manage it better than
his brothers. Ernest was too lazy, and preferred walking at ease with
the monkey on his shoulder, and the more so because it spared him
the trouble of gathering fruit. Jack was too giddy to be trusted alone
on the horse, though he often got up behind his brother, and Francis
still too little to attempt mounting it. Notwithstanding the onagra
was so well broken in for riding, it continued to be very mettlesome
and restive in the shafts, to which we could not inure it; but
occasionally it submitted to our putting a loaded sack or two on its
back; but we could seldom prevail even in this, without Fritz being
seated in front; he would then take them to the house, and thus was
rendered of some general use.
When we reached the oaks Lightfoot was tied to a bush, and we
set actively to work to gather the acorns that had dropped from the
trees. While all were busily employed, the monkey quitted its
master’s shoulder and skipped unperceived into an adjoining bush. It
had been there some time when we heard on that side the loud
cries of birds and flapping of wings, and this assured us a sharp
conflict was going on betwixt master Knips and the inhabitants of
the bushes. I dispatched Ernest to reconnoitre. He went stoutly
towards the place, and in an instant we heard him exclaim, Come
quickly, father! a fine heath-fowl’s nest full of eggs; Mr. Knips, as
usual, wished to make a meal of them; the hen and he are fighting
for it: come quick, Fritz, and take her; I am holding greedy-chops as
well as I can.
Fritz ran up directly, and in a few moments brought out alive the
male and female heath-fowl, both very beautiful; the cock finely
collar’d, similar to one he had killed on a former occasion, not
without much regret on my part. I was rejoiced at this discovery,
and helped my son to prevent their escape by tying their wings and
feet, and holding them while he returned to the bush for the eggs.
And now Ernest came forward driving the monkey before him, and
carrying his hat with the utmost care: he had stuck his girdle full of
narrow sharp-pointed leaves, in shape like a knife-blade, which
reminded me of the production named sword-grass; but I did not
pay much attention, as I was too busily engaged in our egg-hunt,
and considered his decoration as childishness. On coming up to me
he uncovered his hat, and gave it to me in a transport of joy, crying
out, Here, dear father, here are some heath-fowl’s eggs; I found
them in a nest so well concealed under these long leaves that I
should not have observed them had not the hen, in defending
herself against the monkey, scattered them about. I am going to
take them home, they will please my mother; and these leaves will
so amuse Francis, they are like swords, and will be the very thing he
will like for a play-thing. I applauded Ernest’s attention to both, and
I encouraged him and Fritz to be thus ever considerate for the
absent, so as to prove they could never be forgotten. The
kindnesses conferred on those who are separated from us have in
themselves more merit, and are more valued, than those which are
personally received. It was now time to think of moving homeward:
my two sons filled the bags with acorns and put them on Lightfoot;
Fritz mounted, Ernest carried the eggs, I took charge of the hen,
and we proceeded to Falcon’s Stream followed by our train-waggon.
Our good cattle were in such complete subjection that it was only
necessary to speak to them. I remarked Ernest often applying his
ear to the hat which held the eggs, as if he thought the little ones
were near coming forth; I listened also, and observed some shells
already broken and the young protruding: we were overjoyed at our
good luck, and Fritz could not refrain from trotting on briskly to bear
the tidings to his dear mother: but he went rather faster than he
intended on setting out: he had taken a handful of the pointed
leaves with him, which he whisked before the ears and eyes of the
onagra, till the animal was frightened, lost all restraint, and darted
forward with him like a shot, hurrying away bags and rider at such a
rate that we soon lost sight of them. Anxious for his safety, we
followed as fast as possible, though out of sight of him all the way;
but on our arrival at Falcon’s Stream we had the satisfaction of
finding him there in perfect safety. His mother, indeed, had been
somewhat alarmed in seeing him dash in like a thunderbolt, but
firmly seated betwixt the bags on master Lightfoot, who well
deserved his name on this occasion, and who stopped short with
wonderful precision at his stable door. Our first care was to examine
the eggs: the female bird was too frightened and wild to sit upon
them: fortunately we had a hen that was hatching; her eggs were
immediately removed, and the new ones put in their place: the
female heath-fowl was put into the parrot’s cage, and hung up in the
room to accustom it to our society. In less than three days all the
chickens were hatched, they kept close to their foster-mother, and
ate greedily a mixture of sweet acorns bruised in milk, such as we
gave our tame poultry: as they grew up I plucked out the large
feathers of their wings, lest they should naturally take flight; but
they and their real parent gradually became so domesticated, that
they daily accompanied our feathered stock in search of food, and
regularly came back at night to the roost I had prepared for them,
and in which this little new colony of feathered beings seemed to
delight.
 CHAPTER XXXI.
Flax, and the rainy season.

Francis for a short time was highly amused with his sword-leaves,
and then like all children, who are soon tired of their toys, he grew
weary of them, and they were thrown aside. Fritz picked up some of
them that were quite soft and withered; he held up one which was
pliable as a ribband in the hand: My little fellow, said he to his
brother, you can make whips of your sword-grass, take up the leaves
and keep them for this purpose, they will be of use in driving your
goats and sheep. It had been lately decided that it should be the
business of Francis to lead these to pasture.
Well then, help me to make them, said the child. They sat down
together. Francis divided the leaves into long narrow slips, and Fritz
ingeniously platted them into whip-cords. As they were working, I
saw with pleasure the flexibility and strength of the bands; I
examined them more closely, and found they were composed of long
fibres or filaments; and this discovery led me to surmise that this
supposed sword-grass might be a very different thing, and not
improbably the flax-plant of New Zealand, called by naturalists
Chlomidia, and by others Phormion19. This was a valuable discovery
in our situation: I knew how much my wife wished for the
production, and that it was the article she felt most the want of; I
therefore hastened to communicate the intelligence to her, upon
hearing which she expressed the liveliest joy: This, said she, is the
most useful thing you have found; I entreat you, lose not a moment
in searching for more of these leaves, and bring me the most you
can of them; I will make you stockings, shirts, clothes, thread,
ropes.....
In short, give me flax, looms, and frames, and I shall be at no loss
in the employment of it. I could not help smiling at the scope she
gave to her imagination, on the bare mention of flax, though so
much was to be done between the gathering the leaves and having
the cloth she was already sewing in idea. Fritz whispered a word in
Jack’s ear; both went to the stable, and without asking my leave,
one mounted Lightfoot, the other the buffalo, and galloped off
towards the wood so fast that I had no time to call them back; they
were already out of sight: their eagerness to oblige their mother in
this instance pleaded their forgiveness, and I suffered them to go on
without following them, purposing to proceed and bring them back if
they did not soon return.
In waiting for them I conversed with my wife, who pointed out to
me with all the animation and spirit of useful enterprise so natural to
her character, the various machinery I must contrive for spinning and
weaving her flax for the manufactory of cloths, with which she said
she should be able to equip us from head to foot; in speaking of
which, her eyes sparkled with the love of doing good, the purest
kind of joy, and I promised her all she desired of me.
In a quarter of an hour our deserters came back on a full trot, and
I was pleased to see them again; like true hussars, they had foraged
the woods, and heavily loaded their cattle with the precious plant,
which they threw at their mother’s feet with joyful shouts. We could
not blame their abrupt departure. Jack made us laugh in recounting
with his accustomed vivacity and drollery at what a rate he had
trotted his buffalo to keep up with Lightfoot, and how his great
horned horse had thrown him by a side leap; yet that
notwithstanding these, he and his buffalo, as in duty and allegiance
bound, were, as ever, at the entire command of their acknowledged
queen. Well, said I, you shall then all assist her with consummate
diligence in preparations for the work she is about to engage in, and
previously in steeping the flax.
 Fritz.—How is flax prepared, father, and what is meant by steeping
it?
Father.—Steeping flax, or hemp, is exposing it in the open air, by
spreading it on the ground to receive the rain, the wind, and the
dew, in order in a certain degree to liquefy the plant; by this means
the ligneous or cortical parts of the flax are separated with more
ease from the fibrous; a kind of vegetable glue that binds them is
dissolved, and it can then be perfectly cleaned with great facility,
and the parts selected which are fit for spinning.
Fritz.—But may not the natural texture of this part be destroyed
by exposing it so long to wet?
Father.—That certainly may happen when the process is managed
injudiciously, and the flax not duly turned; the risk, however, is not
great, the fibrous part has a peculiar tenacity, which enables it to
resist longer the action of humidity; flax may be even steeped
altogether in water without injury. Many think this the best and
quickest method, and I am of their opinion.
My wife coincided with me, especially in the sultry climate we
inhabited: she therefore proposed to soak the flax in Flamingo
Marsh, and to begin by making up the leaves in bundles, as they do
hemp in Europe. We agreed to her proposal, and joined in this
previous and necessary preparation of the flax during the rest of the
day.
Next morning the ass was put to the small light car, loaded with
bundles of leaves; Francis and the monkey sat on them, and the
remainder of the family gaily followed with shovels and pickaxes. We
stopped at the marsh, divided our large bundles into smaller, which
we placed in the water, pressing them down with stones and leaving
them in this state till our sovereign should direct us to remove and
set them in the sun to dry, and thus render the stems soft and easy
to peel. In the course of this work we noticed with admiration the
instinct of the flamingoes in building their cone-shaped nests above
the level of the marsh, each nest having a recess in the upper part,
in which the eggs are securely deposited, while the contrivance
enables the female to sit with her legs in the water: the nest is of
clay closely cemented, so as to resist all danger from the element till
the young can swim.
A fortnight after, my wife told us the flax was sufficiently steeped.
We then took it out of the water, and spread it on the grass in the
sun, where it dried so well and rapidly that we were able to load it
on our cart the same evening, and carry it to Falcon’s Stream, where
it was put by till we had time to attend further to it, and make
beetles, wheels, reels, carding-combs, &c., as required by our expert
and skilful flax-manufacturer. It was thought best to reserve this task
for the rainy season, and to get ready what would be then necessary
during our confinement within doors. Uninformed as we were as to
the duration of this season, it was highly important to lay in a
competent stock of provisions for ourselves and for all the animals.
Occasional slight showers, the harbingers of winter, had already
come on; the temperature, which hitherto had been warm and
serene, became gloomy and variable; the sky was often darkened
with clouds, the stormy winds were heard, and warned us to avail
ourselves of the favourable moment to collect every thing that would
be wanted.
Our first care was to dig up a full supply of potatoes and yams for
bread, with plenty of cocoa-nuts, and some bags of sweet acorns. It
occurred to us while digging, that the ground being thus opened and
manured with the leaves of plants, we might sow in it to advantage
the remainder of our European corn. Notwithstanding all the
delicacies this stranger land afforded us, the force of habit still
caused us to long for the bread we had been fed with from
childhood: we had not yet laid ourselves out for regular tillage, and I
was inclined to attempt the construction of a plough of some sort as
soon as we had a sufficient stock of corn for sowing. For this time,
therefore, we committed it to the earth with little preparation: the
season, however, was proper for sowing and planting, as the
ensuing rain would moisten and swell the embryo grain, which
otherwise would perish in an arid burning soil. We accordingly
expedited the planting of the various palm trees we had discovered
in our excursions, at Tent House, carefully selecting the smallest and
the youngest. In the environs was formed a large handsome
plantation of sugar canes, so as to have hereafter every thing useful
and agreeable around us, and thus be dispensed from the usual toil
and loss of time in procuring them.
These different occupations kept us several weeks in unremitted
activity of mind and body; our cart was incessantly in motion,
conveying home our winter stock; time was so precious that we
could not even make regular meals, and limited ourselves to bread,
cheese, and fruits, in order to shorten them, to return quickly to our
work, and dispatch it before the bad season should set in.
Unfortunately, the weather changed sooner than we had
expected, and than, with all our care, we could be prepared for;
before we had completed our winter establishment, the rain fell in
such heavy torrents that little Francis, trembling, asked me whether
father Noah’s deluge was coming on again; and I could not myself
refrain from painful apprehension in surmising how we should resist
such a body of water, that seemed to change the whole face of the
country into a perfect lake.
The first thing to be done, and which gave us all sensations of
deep concern, was to remove without delay our aërial abode, and to
fix our residence at the bottom of the tree, between the roots and
under the tarred roof I had erected; for it was no longer possible to
remain above, on account of the furious winds that threatened to
bear us away, and deluged our beds with rain through the large
opening in front, our only protection here being a piece of sail-cloth,
which was soon dripping wet and rent to pieces. In this condition we
were forced to take down our hammocks, mattresses, and every
article that could be injured by the rain; and most fortunate did we
deem ourselves in having made the winding stairs, which sheltered
us during the operation of the removal. The stairs served afterwards
for a kind of lumber-room; we kept all in it we could dispense with,
and most of our culinary vessels, which my wife fetched as she
happened to want them. Our little sheds between the roots,
constructed for the poultry and the cattle, could scarcely contain us
all; and the first days we passed in this manner were painfully
embarrassing, crowded all together, and hardly able to move in
these almost dark recesses, which the fœtid smell from the close-
adjoining animals rendered almost insupportable: in addition, we
were half stifled with smoke whenever we kindled a fire, and
drenched with rain when we opened the doors. For the first time,
since our disaster, we sighed for the comfortable houses of our dear
country:—but what was to be done! we were not there, and losing
our courage and our temper would only increase the evil. I strove to
raise the spirits of my companions, and obviate some of the
inconveniences. The now doubly-precious winding stair was, as I
have said, every way useful to us; the upper part of it was filled with
numerous articles that gave us room below; and as it was lighted
and sheltered by windows, my wife often worked there, seated on a
stair, with her little Francis at her feet. We confined our live-stock to
a smaller number, and gave them a freer current of air, dismissing
from the stalls those animals that from their properties, and being
natives of the country, would be at no loss in providing for
themselves. That we might not lose them altogether, we tied bells
round their necks; Fritz and I sought and drove them in every
evening that they did not spontaneously return. We generally got
wet to the skin and chilled with cold, during the employment, which
induced my wife to contrive for us a kind of clothing more suitable to
the occasion; she took two seamen’s shirts from the chest we had
recovered from the wreck; and then, with some pieces of old coats,
she made us a kind of cloth hoods joined together at the back, and
well formed for covering the head entirely: we melted some elastic
gum, which we spread over the shirts and hoods; and the articles
thus prepared answered every purpose of water-proof overalls, that
were of essential use and comfort to us. Our young rogues were
ready with their derision the first time they saw us in them; but
afterwards they would have been rejoiced to have had the same:
this, however, the reduced state of our gum did not allow, and we
contented ourselves with wearing them in turn, when compelled to
work in the rain, from the bad effects of which they effectually
preserved us.
 As to the smoke, our only remedy was to open the door when we
made a fire; and we did without as much as we could, living on milk
and cheese, and never making a fire but to bake our cakes: we then
availed ourselves of the opportunity to boil a quantity of potatoes
and salt meat enough to last us a number of days. Our dry wood
was also nearly expended, and we thanked Heaven the weather was
not very cold; for had this been the case our other trials would have
much increased. A more serious concern was our not having
provided sufficient hay and leaves for our European cattle, which we
necessarily kept housed to avoid losing them; the cow, the ass, the
sheep, and the goats, the two last of which were increased in
number, required a large quantity of provender, so that we were ere
long forced to give them our potatoes and sweet acorns, which by
the by they found very palatable, and we remarked that they
imparted a delicate flavour to their milk;—the cow, the goats, and
even the sheep, amply supplied us with that precious article:
milking, cleaning the animals and preparing their food, occupied us
most of the morning, after which we were usually employed in
making flour of the manioc root, with which we filled the large
gourds, which were previously placed in rows. The gloom of the
atmosphere and our low windowless habitation sensibly abridged our
daylight; fortunately, we had laid in a huge store of candles, and felt
no want of that article: when darkness obliged us to light up, we got
round the table, when a large taper fixed on a gourd gave us an
excellent light, which enabled my wife to pursue her occupation with
the needle, while I, on my part, was forming a journal and recording
what the reader has perused of the narrative of our shipwreck and
residence in this island, assisted from time to time by my sons and
their admirable mother, who did not cease to remind me of various
incidents belonging to the story. To Ernest, who wrote a fine hand,
was intrusted the care of writing off my pages in a clear legible
character; Fritz and Jack amused themselves by drawing from
memory the plants and animals which had most struck their
observation; while one and all contributed to teach little Francis to
read and write: we concluded the day with a devotional reading in
the Holy Bible, performed by each in turn, and we then retired to
rest, happy in ourselves, and in the innocent and peaceful course of
our existence. Our kind and faithful steward often surprised us
agreeably on our return from looking after the cattle, by lighting up
a faggot of dried bamboo, and quickly roasting by the clear and
fervent heat it produced, a chicken, pigeon, duck, or penguin from
our poultry-yard, or some of the thrushes we had preserved in
butter, which were excellent, and welcomed as a treat to reward
extraordinary toil. Every four or five days the kind creature made us
new fresh butter in the gourd-churn; and this with some deliciously
fragrant honey spread on our manioc cakes, formed a collation that
would have raised the envy of European epicures. These unexpected
regales represented to our grateful hearts so many little festivals, the
generous intention of which made us forget our bad
accommodations and confinement.
The fragments of our meals belonged in right to our domestic
animals, as part of the family. We had now four dogs, the young
jackal, the eagle, and the monkey, to feed; they relied with just
confidence on the kindness of their respective masters, who
certainly would have deprived themselves to supply the wants of
their helpless dependents. Francis had taken under his mighty
protection the two little bull-dogs; my wife Ponto, and I the brave
Turk:—thus each had his attendant, of which he took care, and no
one was dispensed from the offices of tenderness and vigilance. If
the buffalo, the onagra, and pig had not found sustenance abroad,
they must have been killed or starved, and that would have given us
much pain. In the course of these discomforts it was unanimously
resolved on, that we would not pass another rainy season exposed
to the same evils; even my beloved consort, who felt such a
predilection for the abode at Falcon’s Stream, was frequently a little
ruffled and out of temper with our inconvenient situation, and
insisted more than any of us on the propriety of building elsewhere a
more spacious winter residence: she wished, however, to return to
our castle in the tree every summer, and we all joined with her in
that desire. The choice of a fresh abode now engrossed our
attention, and Fritz in the midst of consultation came forward
triumphantly with a book he had found in the bottom of our clothes’
chest. Here, said he, is our best counsellor and model, Robinson
Crusoe; since Heaven has destined us to a similar fate, whom better
can we consult? as far as I remember, he cut himself an habitation
out of the solid rock: let us see how he proceeded; we will do the
same and with greater ease, for he was alone; we are six in number,
and four of us able to work. Well spoken, son, said I: this activity
and courage give me pleasure; let us then strive to be as ingenious
as Robinson Crusoe.
And why not? observed Jack—Have we not an island, rocks, and
tools from abroad as good as he had, and, as brother Fritz says,
more hands to use them?
We assembled, and read the famous history with an ardent
interest; it seemed though so familiar, quite new to us: we entered
earnestly into every detail and derived considerable information from
it, and never failed to feel lively gratitude towards God who had
rescued us all together, and not permitted one only of us to be cast
a solitary being on the island. The occurrence of this thought
produced an overwhelming sense of affection among us, and we
could not refrain from throwing ourselves into each others arms,
embracing repeatedly, and the pathetic scene ended in mutual
congratulations.
Francis repeated his wish to have a Man Friday; Fritz thought it
better to be without such a companion, and to have no savages to
contend with. Jack was for the savages, warfare and encounters.
The final result of our deliberations was to go and survey the rocks
round Tent-House, and to examine whether any of them could be
excavated for our purpose.
Our last job for the winter, undertaken at my wife’s solicitation,
was a beetle for her flax and some carding-combs. I filed large nails
till they were even, round, and pointed; I fixed them at equal
distances in a sheet of tin, and raised the sides of it like a box; I
then poured melted lead between the nails and the sides, to give
firmness to their points, which came out four inches. I nailed this tin
on a board, and the machine was fit for work. My wife was impatient
to use it; and the drying, peeling, and spinning her flax, became
from this time a source of inexhaustible delight.
 CHAPTER XXXII.
Spring;—spinning;—salt mine.

I can hardly describe our joy when, after many tedious and gloomy
weeks of rain, the sky began to brighten, the sun to dart its benign
rays on the humid earth, the winds to be lulled, and the state of the
air became mild and serene. We issued from our dreary hovels with
joyful shouts, and walked round our habitation breathing the
enlivening balmy ether, while our eyes were regaled with the
beauteous verdure beginning to shoot forth on every side. Reviving
nature opened her arms, every creature seemed reanimated, and we
felt the genial influence of that glorious luminary which had been so
long concealed from our sight, now returned like a friend who has
been absent, to bring us back blessings and delight. We rapidly
forgot in new sensations the embarrasments and weary hours of the
wet season, and with jocund, hopeful hearts, looked forward to the
toils of summer as enviable amusements.
The vegetation of our plantation of trees was rapidly advancing;
the seed we had thrown into the ground was sprouting in slender
blades that waved luxuriantly; a pleasing tender foliage adorned the
trees; the earth was enamelled with an infinite variety of flowers,
whose agreeable tints diversified the verdure of the meadows.
Odorous exhalations were diffused through the atmosphere; the
song of birds was heard around; they were seen between the leaves
joyfully fluttering from branch to branch; their various forms and
brilliant plumage heightened this delightful picture of the most
beautiful spring, and we were at once struck with wonder and
penetrated with gratitude towards the Creator of so many beauties.
Under these impressions we celebrated the ensuing Sunday in the
open air, and with stronger emotions of piety than we had hitherto
felt on the fertile shores upon which we had been so miraculously
saved and fostered. The blessings which surrounded us were ample
compensation for some uneasy moments which had occasionally
intervened, and our hearts, filled with fresh zeal, were resolved to be
resigned, if it should be the will of God, to pass the residue of our
days in this solitude with serenity of soul and every due exertion.
The force of paternal feelings, no doubt, made me sometimes form
other wishes for my children; but these I buried in my own breast,
for fear of disturbing their tranquillity: but if I secretly indulged a
desire for some event that might prolong and even increase their
happiness, I nevertheless wholly submitted all to the Divine will, the
manifestation of which I awaited in becoming thankfulness and
patience.
Our summer occupations commenced by arranging and thoroughly
cleaning Falcon’s Nest, the order and neatness of which the rain and
dead leaves blown by the wind had disturbed: in other respects,
however, it was not injured, and in a few days we rendered it
completely fit for our reception; the stairs were cleared, the rooms
between the roots re-occupied, and we were left with leisure to
proceed to other employments. My wife lost not a moment in
resuming the process of her flax concern. Our sons hastened to lead
the cattle to the fresh pastures, already dried by the sun; whilst it
was my task to carry the bundles of flax into the open air, whereby
heaping stones together I contrived an oven sufficiently commodious
to dry it well. The same evening we all set to work to peel, and
afterwards to beat it and strip off the bark, and lastly to comb it with
my carding machine, which fully answered the purpose. I took this
somewhat laborious task on myself, and drew out such distaffs full of
long soft flax ready for spinning, that my enraptured wife ran to
embrace me, to express her heartfelt acknowledgement, requesting
me to make her a wheel without delay, that she might enter upon
her favourite work.
 At an earlier period of my life I had practised turnery for my
amusement; now, however, I was unfortunately destitute of the
requisite utensils; but as I had not forgotten the arrangement and
component parts of a spinning-wheel and reel, I by repeated
endeavours found means to accomplish those two machines to her
satisfaction; and she fell so eagerly to spinning, as to allow herself
no leisure even for a walk, and scarcely time to dress our dinners:
nothing so much delighted her as to be left with her little boy, whom
she employed to reel as fast as she could spin, and sometimes the
other three were also engaged in turns at the wheel, to forward her
business whilst she was occupied in culinary offices; but not one of
them was found so tractable as the cool-tempered quiet Ernest, who
preferred this to more laborous exertions, though such was our want
of linen and clothes, that we ought all readily and even eagerly to
have joined in procuring them; but our excursions, and the
necessary liberty they involved, were more agreeable to us than this
female occupation. Our first visit was to Tent-House, as we were
anxious to ascertain the ravages of winter there, and we found them
much more considerable than at Falcon’s Stream, and even dreadful:
the tempest and rain had beaten down the tent, carried away a part
of the sail-cloth, and made such havoc amongst our provisions, that
by far the largest portion of them was spotted with mildew, and the
remainder could be only saved by drying them instantly. Luckily, our
handsome pinnace had been for the most part spared; it was still at
anchor, ready to serve us in case of need; but our tub-boat was in
too shattered a state to be of any further service.
In looking over the stores we were grieved to find the gunpowder
most damaged, of which I had left three barrels in the tent instead
of placing them in a more sheltered situation in the cavity of the
rock. The contents of two were rendered wholly useless. I thought
myself fortunate on finding the remaining one in tolerable condition,
and derived from this great and irreparable loss a cogent motive to
fix upon winter quarters where our stores and wealth would not be
exposed to such cruel dilapidations.
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