Experience Seamless Full Ebook Downloads for Every Genre at textbookfull.

com

Your Passport to a Career in Bioinformatics 2nd

Edition Prashanth N. Suravajhala

https://textbookfull.com/product/your-passport-to-a-career-
in-bioinformatics-2nd-edition-prashanth-n-suravajhala/

OR CLICK BUTTON

DOWNLOAD NOW

Explore and download more ebook at https://textbookfull.com

 Recommended digital products (PDF, EPUB, MOBI) that
you can download immediately if you are interested.

Your Passport to Gifted Education 1st Edition Monita

Leavitt (Auth.)

https://textbookfull.com/product/your-passport-to-gifted-
education-1st-edition-monita-leavitt-auth/

textboxfull.com

What Color Is Your Parachute 2021 Your Guide to a Lifetime

of Meaningful Work and Career Success 9781984857880
2021st Edition Richard N. Bolles
https://textbookfull.com/product/what-color-is-your-
parachute-2021-your-guide-to-a-lifetime-of-meaningful-work-and-career-
success-9781984857880-2021st-edition-richard-n-bolles/
textboxfull.com

You Majored in What Designing Your Path from College to

Career 1st Edition Katharine Brooks

https://textbookfull.com/product/you-majored-in-what-designing-your-
path-from-college-to-career-1st-edition-katharine-brooks/

textboxfull.com

Passport to Successful ICU Discharge Carole Boulanger

https://textbookfull.com/product/passport-to-successful-icu-discharge-
carole-boulanger/

textboxfull.com
The Heart of a Leader Fifty Two Emotional Intelligence
Insights to Advance Your Career Harper

https://textbookfull.com/product/the-heart-of-a-leader-fifty-two-
emotional-intelligence-insights-to-advance-your-career-harper/

textboxfull.com

Building Your Academic Research Digital Identity: A Step-

Wise Guide to Cultivating Your Academic Research Career
Online 1st Edition Margaret Rush Dreker
https://textbookfull.com/product/building-your-academic-research-
digital-identity-a-step-wise-guide-to-cultivating-your-academic-
research-career-online-1st-edition-margaret-rush-dreker/
textboxfull.com

The Squiggly Career: Ditch the ladder, discover

opportunity, design your career: The five skills you need
to succeed in work today First Edition Helen Tupper
https://textbookfull.com/product/the-squiggly-career-ditch-the-ladder-
discover-opportunity-design-your-career-the-five-skills-you-need-to-
succeed-in-work-today-first-edition-helen-tupper/
textboxfull.com

Back in the game why concussion doesn t have to end your

athletic career 1st Edition Gerstner

https://textbookfull.com/product/back-in-the-game-why-concussion-
doesn-t-have-to-end-your-athletic-career-1st-edition-gerstner/

textboxfull.com

Principles of Product Management: How to Land a PM Job and

Launch Your Product Career 1st Edition Peter Yang

https://textbookfull.com/product/principles-of-product-management-how-
to-land-a-pm-job-and-launch-your-product-career-1st-edition-peter-
yang/
textboxfull.com
Prashanth N. Suravajhala Editor

Your Passport
to a Career in
Bioinformatics
Second Edition
Your Passport to a Career in Bioinformatics
Prashanth N. Suravajhala
Editor

Your Passport to a Career

in Bioinformatics
Second Edition
Editor
Prashanth N. Suravajhala
Department of Biotechnology
and Bioinformatics
Birla Institute of Scientific Research
Jaipur, India
Bioclues Organization
Hyderabad, India

ISBN 978-981-15-9543-1 ISBN 978-981-15-9544-8 (eBook)

https://doi.org/10.1007/978-981-15-9544-8

© Springer Nature Singapore Pte Ltd. 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the
material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
To my Mother Nirmala Sastry
Foreword

When I first heard about the field of bioinformatics, I was a university senior
majoring in chemistry. It was 1995, and my intention at the time was to focus on
the application of chemistry in the life sciences. In fact, in those days I was interested
in any field of science or engineering that could be applied to biology. But, when it
came time to select a project for my senior thesis, I was asked by my thesis adviser if
I had an interest in computers. Certainly, I did. I had a year of computer science
courses under my belt, but I also had an avid interest in computers as a hobby—I
wrote my first BASIC program circa 1981 on a friend’s Atari800. And, so my
adviser proceeded to tell me that there is this nascent field called “bioinformatics,”
which is a hybrid of computer science and biology. I immediately fell in love with
the idea that I could combine a professional interest of mine with a personal one.
And, from then on, even through graduate school, all of my research projects
involved programming. Not one required that I stand at a bench with a micropipette,
as I knew I would be doing as a biochemist. Of course, it did not go over so well with
many of the professors back then that a student would pursue a degree in either
biochemistry or biology with a purely computational project. In the 1990s, there
were just a handful of degree programs in bioinformatics in the whole world—one of
them halfway around the world from where I lived. But I limited my own geograph-
ical options, and it seemed that my only choice was to pursue a graduate degree in
“traditional” biochemistry and find an adviser and laboratory group that had an
interest in performing computational analyses on their data.
Fortunately for aspiring scientists today, there are many straightforward ways to
enter the field of bioinformatics. To that point, there are scores of degree programs
throughout the world—many of them online degrees. And, there are other ways to
further one’s own career as a bioinformatics practitioner. For one, there is the
Bioinformatics.Org website, of which I am the founder, with Prashanth Suravajhala
among the directors. Prash also founded Bioclues.org and has been active in

vii
viii Foreword

mentoring students online regarding their academic projects in bioinformatics. It is

because of this experience of his that I think you will be enlightened by the insight
that Prash shares within these pages.

Bioinformatics.Org, Hudson, MA, USA J. W. Bizzaro

Acknowledgments

I thank Messers Springer, Bhavik Sawhney, Beracah John Martyn and Camilya
Anitta for agreeing to my request to reconsider the revised edition of this book and
for their consistent help in proofreading. Aninda Bose and Chandra Shekhar of
Springer who have supported me all through the making of the first edition of the
book have played a major role. Although the cartoons and illustrations were ideated
by me, full credits to Partha Paul for bringing life to them.
My sincere obeisances to my revered Guru Maa Bijaya for her grace and
blessings. My sincere gratitude goes to my mentees without whose thoughts this
book would not have been here today. Likewise, I owe appreciation to my wife
Renuka and my daughters Bhavya and Nirmala who always stood by me.
My peers in Bioclues.org and bioinformatics.org, ex-colleagues, and researchers
in India, Denmark, the United States, and Japan, countless “e-colleagues,” also
contributed to my discussions. I sincerely thank Cox Murray, Jeff Bizzaro, Madhan
Mohan, and Pawan Dhar who were generous enough to have responded to the
questionnaire. My grandparents—Shri D. S. Sastry and D. S. R. Murthy are always
remembered with fond love and affection. They have helped me in imparting clarity,
coherence, and brevity to the text.
Finally, the book would not have come into good shape without the help of
contributions from various authors across four continents, Springer reviewers,
friends, and well-wishers, but not the least the author sincerely thanks the Springer
typesetting team, Messers Nalini Gyaneshwar, Kamiya Khatter et al. for bringing the
manuscripts in shape.

ix
Prologue

Today, we define success by publicity and bank accounts. But that is not really
success at all. Do not believe the hype. Success is ephemeral. You have to define it
yourself.
Chris North
Most people would succeed in small things if they were not troubled with great
ambitions.
Henry Wadsworth Longfellow
Any new word invites inquiry, excitement, and sometimes disdain and so was
bioinformatics, at least in developing countries. Theoretical bioinformatics, although
born in the 1980s, has flourished ever since, as many new academic and empirical
developments with focal point on wet-lab research confirm. Bioinformatics is now
regarded as a tool but fantasized as a familiar science even by few scientists who
have had a track record of early career building. With research on bioinformatics
mushrooming, both theoretical and wet-lab-based bioinformatics-aided works are
often deemed very procedural and paraphernalia that these are not easily accessible
to those who want to use the “tools for biology.” Additionally, the career-driven
paths using bioinformatics is tacit by the fact that one needs to attend to earn
programming skills which is not always the case. This book aims to be an interface
between those who aim for bioinformatics and apply research with a focus on Q and
A on career growth. A great saying goes “If you want more, you have to require
more from yourself.” This also applies to bioinformatics. Happy reading!

Department of Biotechnology and Prashanth N. Suravajhala

Bioinformatics, Birla Institute of
Scientific Research, Jaipur, India

xi
Contents

1 Whither Bioinformatics? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Prashanth N. Suravajhala
2 Ten Reasons One Should Take Bioinformatics as a Career . . . . . . . 25
Prashanth N. Suravajhala
3 Developing Bioinformatics Skills . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Prashanth N. Suravajhala
4 The Esoteric of Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Prashanth N. Suravajhala
5 Common Minimum Standards: A Syllabus for Bioinformatics
Practitioners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Prashanth N. Suravajhala
6 Colloquial Group Discussion on Bioinformatics:
Grand Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Prashanth N. Suravajhala
7 The Bioinforma “TICKS”: Frequently Asked Questions . . . . . . . . . 69
Prashanth N. Suravajhala
8 Undergraduate Education in Bioinformatics—Progress and Lessons
Learnt from an Engineering Degree . . . . . . . . . . . . . . . . . . . . . . . . 73
Bruno A. Gaeta
9 Engineering Minds for Biologists . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Alfredo Benso, Stefano Di Carlo, and Gianfranco Politano
10 Design Bioinformatics Curriculum Guidelines: Perspectives . . . . . . 91
Qanita Bani Baker and Maryam S. Nuser
11 Machine Learning for Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . 103
Harshita Bhargava, Amita Sharma, and Jayaraman K. Valadi

xiii
xiv Contents

Bioinformatics Cross Word . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
About the Editor

Prashanth N. Suravajhala is currently working as a

Senior Scientist at Birla Institute of Scientific Research
(BISR), Jaipur. Previously, he has obtained a Ph.D. in
Systems Biology. He has been involved in various pro-
jects specific to immunomodulatory/metabolic diseases
and cancer biology. He has identified candidate genes
using in silico approaches and provided a standard clas-
sification and scoring scheme for characterizing hypo-
thetical proteins in vitro, specifically those that are
targeted to mitochondria. More recently, his interests
have expanded to transcriptomic profiling, functional
proteomics, and molecular genetics of cancer/diseasome
approaches linked to evolutionary aspects in under-
standing functional aspects of regulatory genes,
epigenomic profiling, miRNAs, and long noncoding
RNAs. He is a founder of Bioclues.org, a not-for-profit
organization through which he mentors bioinformatics
graduates

xv
Chapter 1
Whither Bioinformatics?

Prashanth N. Suravajhala

Ever since the word “Theoretical Biology” was coined by Paulien Hogeweg in1978,
bioinformatics, the current word has steadfastly come into existence with many
biologists taking a leaf out of this discipline. Researchers by now know that bioinfor-
matics is a mere tool, whereas its sister concern, computational biology, is deemed as a
discipline. With bioinformatics burgeoning in the late 1990s, we relate the commence-
ment of data deluge to the animistic knowledge that bioinformatics has brought in,
lessening the scale of experimentation. Authentic bioinformatics, however, will not
gain significant interest for researchers, at least until the wet laboratory biologists take
a leap forward in acclimatizing the split half-term in bioinformatics. The figure of
dogmas is pivotal in bringing the collaboration between biologists and cross-
disciplinarians across biology as the event of dogmas in turn has introduced a plethora
of new relationships between scientific studies and molecular biology. In effect,
researchers have asked several questions on specialized mechanisms, if any that may
be discovered in the advent of bioinformatical knowledge. This collaborative knowl-
edge owes its impetus to the differentiation of independent eccentric science, namely,
systems biology (SB). So, to ask whither bioinformatics into the enunciation and
practice of the bioinformatical tools and scientific methods is a candid query.
Bioinformatics, since ages, has created a process of reasoning that was certainly
not dependent on biology alone. Prior notions of intelligent algorithms clubbed with
statisticians’ skills, IT scientists’ inclination, physicists’ predictions, chemists’ cor-
ner, and mathematicians’ mind are a necessity to perform bioinformatics research.
Not all disciplines can be made up by an individual alone but need unicentric efforts
to meet the goals to derive bioinformatics knowledge. For example, the next

P. N. Suravajhala (*)
Department of Biotechnology and Bioinformatics, Birla Institute of Scientific Research, Jaipur,
India
Bioclues Organization, Hyderabad, India
e-mail: [email protected]; http://bioclues.org

© Springer Nature Singapore Pte Ltd. 2021 1

P. N. Suravajhala (ed.), Your Passport to a Career in Bioinformatics,
https://doi.org/10.1007/978-981-15-9544-8_1
2 P. N. Suravajhala

generation sequencing (NGS) technologies have enabled non-sanger based sequenc-

ing technologies with unprecedented speed, thereby enabling novel biological appli-
cations. However, before bioinformatics and NGS stepped into the limelight, it must
be noted that the NGS had overcome torpor in the field with the help of several cross-
disciplinarians. It would never have been easy to stir up this understanding without
the rapid involvement of the multifaceted scientists who have transformed biology as
a whole. This obviously has the advantages of building up cross-disciplines, thereby
deepening the knowledge curve between eccentric biology and information science,
the latter constantly teaming up with the former to signify its discoveries with
dogmas.
The greatest challenge facing the molecular biology community today is to make
sense of the wealth of data that have been produced by genome sequencing projects.
Conventional biology research was deemed always to be in the laboratory until the
data deluge and explosion of genomic scale in the late 1990s. Thus, we are in an age
of computing-to-research process. There are two different challenges one would
pose: (1) sequence generation and (2) ensuring storage of the plethora of sequences
generated in the laboratory with specific understanding and investigation using
computers and artificial intelligence. That said, understanding the biology of an
organism is a trivial issue as there are a number of focused research areas at different
levels of “omics”-es, namely, genomics, proteomics, functomics, transcriptomics,
need to being carried out at different levels. One of the foremost challenges today is
to ensure that such data are efficiently stored, used through three forms of Es—
extracting, envisaging, and elucidating this mass of data. A meaningful interpreta-
tion of such data must be done before one analyzes the complete volume for
interpreting it or what we call “annotating” manually. In conclusion, discerning
the function using computer tools must be the focus so as to have meaningful
biological information explained.
The journey of transcriptomics starts with the discovery of ribonucleic acids in
1869 followed by their role in protein synthesis and as a catalyst in various
biochemical reactions. However, the term transcriptomics first appeared in 1998 in
the scientific literature (https://en.wikipedia.org/wiki/Transcriptomics_technologies)
concurrent with different “omics” terminologies. Different “omes” and their respec-
tive descriptions are summarized in Table 1.1.
Why is bioinformatics interesting? All the central biological processes revolve
around bioinformatics tools that need to be developed with a possible leeway in
understanding the sequence–structure–function relationship (see Fig. 1.1). DNA
sequence determines the protein sequence which determines structure and function.
Why is it that we end up with protein as a determiner for every analysis? The
simplest answer is that we would have less noise when we deal with protein
sequences wherein, we deal with 20 odd amino acids to narrate results unlike the
several compositions of four bases, namely, ATGC compendium of six reading
frames translating into amino acids. This integration of information making up
biological processes would allow us to understand the complete repertoire of the
biology of organisms. However, the challenge faced by the biology community,
especially on the inordinate data, is more from the umpteen genome sequencing
projects. Traditionally, wet laboratory biologists carry experimental work even as
1 Whither Bioinformatics? 3

Table 1.1 Components defining different ‘omics’ technologies. The word ‘ome’ refers to ‘many’
or ‘monies.’ For example, genomes indicate the study of many genes
‘Omes’ Description
Genome The full complement of genetic information both coding and noncoding in an
organism
Proteome The complete set of proteins expressed by the genome in an organism
Transcriptome The population of mRNA transcripts in the cell, weighted by their expression
levels as transcripts copy number
Metabolome The quantitative complement of all the small molecules present in a cell in a
specific physiological state
Interactome Product of interactions between all macromolecules in a cell
Phenome Qualitative identification of the form and function derived from genes, but
lacking a quantitative, integrative definition
Glycome The population of carbohydrate molecules in the cell
Translatome The population of mRNA transcripts in the cell, weighted by their expression
levels as protein products
Regulome Genome wide regulatory network of the cell
Operome The characterization of proteins with unknown biological function
Synthetome The population of the synthetic gene products
Hypothome Interactome of hypothetical proteins

Fig. 1.1 An overview of the dogma of molecular biology with known specialized and unknown
mechanisms/flows. (Image courtesy: Daniel Horspool)

the huge increase in the scale of data being produced from time to time could be
better facilitated by in silico analysis. With the help of high-performance computing
(HPC), sequences generated can be sporadic and further analyzed. Nevertheless,
given the fact that molecular biology of a system is very complex, understanding and
disseminating the information is to carried out at different levels using the “omes”
4 P. N. Suravajhala

including the genome, proteome, transcriptome, and metabolome levels. There is a

need for researchers, especially from the wet laboratory community, to herald
bioinformatics indefatigably both in academia and industry.
What discoveries interest researchers? Looking at the dogmas, it is still not clear
whether or not a protein can replicate another protein or a DNA can be obtained by
direct reverse translation. It would be intriguing to understand if these could really
happen in an organism. Can a genomic repertoire take shape in understanding the
dogmas bottom-up? There is an aspiration but puny hope that bioinformatics can
handle this. For example, protein–protein interactions (PPI) play a huge role in
understanding the function of proteins. Various bioinformatics tools have been
developed that allow researchers to compare proteins. Such comparative studies
using algorithms such as BLAST (Altschul et al. 1990, 2005; Alstchul 1991) and
other tools were carried out to distinguish unique proteins from paralogs, which later
might have resulted from gene duplication events. The genomes sequenced so far
were helpful in predicting not only evolutionary relationships but also identified
function for the genes through functional genomics (Link et al. 1997a, b). The in
silico methods such as homology search, presence of motifs, domains and signature
sequences, orthology mapping, and radiation hybrid transcript mapping (Avner et al.
2001) are available for descriptive predictions of proteins with known (and some-
times unknown) function. However, these employed methods possibly might have a
lot of false positives unless in vitro and/or in vivo experiments are followed to
validate them. Moreover, these methods do not reveal a predicted function of
hypothetical proteins (HP), thus making predictions more insignificant. Although
all these methods are being employed by researchers, screening of HPs for novel
translatable candidates is not often used and the researcher repeatedly performs the
screening with laborious wet laboratory experiments. Furthermore, the proteins
whose function remains unknown (i.e., those that remain hypothetical) and that are
targeted to different organelles, especially mitochondria, could be important.
Many protein sequences contain motifs or short signature sequences called
equivalogs (Haft et al. 2003), which are conserved in several organisms. These are
a set of homologous proteins conserved since their last common ancestor with
respect to function (Pearl et al. 2002). Some of these proteins might have a chance
to be duplicated in organisms. It is therefore necessary to understand the genomic
context of such proteins. An example of equivalog model is TIGR00658, identified
as ornithine carbamoyltransferase. However, this enzyme is also known to act in an
arginine biosynthesis pathway from ornithine (TIGR00032 and TIGR00838) in
Yersinia pestis and arginine degradation (TIGR00746 and TIGR01078) in Strepto-
coccus pneumoniae. The TIGRFAMs models, a TIGR family database, include
equivalog models that have been used extensively in genome annotation. In addition,
proteins with weak sequence similarity and no relevant structural homologies
usually do not have known cellular function; such proteins are discarded from
well-known proteins. When annotating proteins, a new molecular role for known
cellular function is carefully addressed and curated. In many cases, there are
numerous proteins that fall under domains whose functions are essentially known
but they have no genuine role played in genomes. In addition, when annotating, if a
1 Whither Bioinformatics? 5

Known Unknowns

Knowns KK KU

UK UU
Unknowns

Fig. 1.2 The importance of Known Unknowns aliased “hypothetical genes” in the genome,
illustrated in the form of a checkerboard. The Known is acronymed “K” while the Unknown
“U.” Apparently, we seldom find “UU”s as it is a misnomer here. Unless the genome is sequenced,
we find genes evaluating and devaluating

reference genome is considered besides comparing sequences in UniGene database

(see web references) with selected protein reference sequences, the alignments
would possibly suggest the function of a gene and finally, the possibility of anno-
tating the protein as the hypothetical would be reduced. For example, many proteins
in humans have been named as some repeat domains (for example, accession
#CAB98209.1) maintaining homology to some known domains and all of them
fall under a large category of domains. This does not necessarily mean that all these
proteins makeup a function. There are also some instances of some proteins already
similar to some organisms not showing up the function, although possibly studied
from in silico and a few wet laboratory studies. Two such examples, one from
eukaryotes and the other from bacteria, are discussed in what follows:
1. The Ankyrin repeat domain 16 (Ankrd16) has protein similarities in mice with
100% (Accession #NP_796242.1), humans with 85.7% (Accession
#NP_061919.1), Xenopus with 71.1% (Accession #NP_001088685.1), and
Danio rerio with 66.6% (Accession #NP_001017563.2). This annotation as
revealed by GenBank and UniGene reference might have an update at a later
point of time when new orthologs as identified from other metazoan sequences
keep adding up to the annotation.
2. In bacteria, the proline proline and glutamic acid (PPE) and the proline glutamic
acid (PE) gene families comprise many unique genes, some of them novel and
labeled as hypothetical. Of them, many are known to be pseudogenes (Marri et al.
2006). The 10% of the coding DNA of Mycobacterium tuberculosis constitutes
PE and PPE family genes and is involved in gene expression upon infection of
macrophages, some of them as antigens mediating role in the pathogenesis or
virulence. These were characterized while the expression levels and the functions
of select PE/PPE family genes during various phases of infection (latent/mild/
hypoxic) with M. tuberculosis (Kim et al. 2008) were studied (Fig. 1.2).
The aforementioned examples discussed are all resultant of the explosion of
bioinformatics tools during the last three decades. Have bioinformatics technologies
6 P. N. Suravajhala

Table 1.2 Pros and cons of different methods in annotating sequence

1. Sequence-based methods
Pros: Most known/reliable method
Cons: BLAST hits are electronically annotated and turn out to be false positives
2. Structure-based methods
Pros: Based on active site characterization/global fold similarity
Cons: Free energy minima always need to be set/obligation
3. Associated-based methods
Pros: Based on the domain/phylogenetic profiles
Cons: Lack of conserved proximity does not indicate a lack of functional association
4. Proteomics-based methods
Pros: Based on protein interaction domains. Gaps or holes in the known pathway can be
assigned. Function awaits a protein to be characterized
Cons: Lots of false positives

revolutionized genomics and proteomics? Well, there has been a focus on molecular
medicine which paved the way for establishing intervention and treatment of well-
known diseases to proactive prediction and prevention of disease risk. These
approaches should really require new informatics systems that will link large-scale
databanks and special programs for data mining and retrieval in bioinformatics and
chemoinformatics. All the wet laboratories should be able to provide a platform for
powerful new molecular diagnostic tools along with multianalyte assays for expres-
sion of genes and proteins in different patterns of diseases. With researchers scaling
the ladder of bioinformatics progress by leaps and bounds, there is a need for an
enhanced understanding of the interactions in a system (organism). What are the
components that interact with each other? What is the outcome of such interactions?
Do interactions alone provide us the functional decipherment? Should we just be
sufficed with the progress made on say, cures for diseases by the year 2050? Should
we reach a consensus on the combination of tools, namely, rapid and inexpensive
DNA sequencing technologies, HapMap project, dollar one genome (DOG), and so
on? We hope that this will let us understand precisely how bioinformatics transits
from research to vocation and avocation (Table 1.2).

1.1 Bioinformatics “Aging” in Systems Biology

Systems biology has gained a lot of attention over the years. Of late, biologists have
been actively engaged in this discipline in different forms when molecular biology is
merged with multi-context disciplines. During this process, SB ran into several
definitions. To answer what is a system: We could think of multiple organelles
existing in our human body as we use components to describe entities in a system.
As we integrate various vehicular components to construct a vehicle, we describe
components such as organelles to makeup a living system. The biology of the system
1 Whither Bioinformatics? 7

Table 1.3 Timeline eventing important spheres in bioinformatics

• 1859 Charles Darwin’s “origin of species”
• 1944 Avery, MacLeod, McCarty: DNA is the genetic material
• 1953 Structure of DNA
• 1955 Complete sequencing of insulin
• 1988 National Center for Biotechnology Information (NCBI) founded
• 1988 Sanger Centre, Hinxton, UK
• 1994 EMBL European Bioinformatics Institute, Hinxton, UK
• 1995 First bacterial genomes completely sequenced
• 1996 Yeast genome completely sequenced
• 1999 Fly genome completely sequenced
• 2000 bioinformatics.org and opensource
• 2001 Human genome and bioinformatics ****Systems Biology****
• 2002–2004 Umpteen genomes sequenced
• 2005 EVOLUTION in terms of bioinformatics as a breakthrough
• 2007 Personal genomics*person “omics”
• 2008 The hypothetical proteins and orphan genes???
• 2011 Predictive biology approaches
• 2012 Next Generation Sequencing burgeons
• 2014 Oxford Nanopore changed the pace of NGS with its first product
• 2016–2019 ScRNA-Seq and spatial genomics era

is called systems biology. Every system has an effect on its environment and so does
the components in a system, even as the components entitled to SB include genes,
proteins, metabolites, and enzymes as minor entities, while cells, tissues, organelles,
and organs as major components. Hence, interactions among the components would
be interesting to value SB. While a system could have many organelles and the
components that makeup the flow of a system, they are bound to interact with one
another. For example, enzymes, proteins, metabolites, genes, DNA, and functional
protein domains are known to interact with each other. Integrating all the interactions
of components indicates: Which survives (and competes) the best while the ultimate
goal of SB is to exploit the interplay among the components. From a reductionism’s
point of view, researchers define SB based on whether the components in a system
are interacting with each other, mutations arising and falling, proteins evaluating and
devaluating, strains adapting and unfitting in the environment, and some genes if lost
and found (Table 1.3).
8 P. N. Suravajhala

1.2 Defining Systems Biology Through Omics: The Two

Paradigms

Is systems biology (SB) all about the genes making up the proteins and how the
components processing in a system interact with each other? The fields of omics in
the recent past have believably revolutionized biomedicine and by far means there
needs to be a focus on change in defining these upcoming omics-es. Huang S’s
classification of SB has yielded the loose and the apparent but broadened definitions
from the dynamics and reductions approach (Huang 2004). The dynamicity of SB is
based on a pure level where the system is based on models and networks: Be it
quantitative or qualitative, whereas the reductionism defines SB based on the high-
throughput methods involving different molecular biology techniques. Overall, the
loose definition applies to projects exploring individual biological networks, while
the broadened but still “derivative” definition is the outgrowth of theoretical models
along with systems theory across interdisciplinary sciences such as engineering,
mathematics, statistics, artificial intelligence, and so forth. However, many authors
(Tracy 2008; Cornish-Bowden et al. 2007; Huang and Wikswo 2006; Strömbäck
et al. 2006; Bruggeman and Westerhoff 2007) have deliberated that the concept of
the gene resulting in omics has begun to outlive its usefulness while they felt that the
SB could be projected into several dimensions keeping in view the multifaceted
systems’ complexity of living organisms (Ideker and Hood 2019). With SB matur-
ing, researchers have started proposing an alternative means to define gene based on
a richer explanation: Genetic functor, or genitor, a sweeping extension of the
classical genotype/phenotype paradigm that describes the “functional” gene (Fox
Keller and Harel 2007). Thus, we could understand the dynamic behaviors of
molecular associations implicitly known from various methods and technologies
integrating one or more of the SB data:
Overall, SB can be envisaged keeping in view the following points:
1. Systems biology is conceptualized in terms of PPI. The interplay between
components in systems is exploited between protein–protein, domain–domain,
DNA–DNA as a whole, or even a protein–DNA.
2. The interactions among the components are better explained in such a way that
what is in theory need not fit practically implicating that a hypothesis-driven
approach need not always be experimental (biological) driven.
3. With some answers to questions like if there are interactions known, we can take a
measure of unknown interactions in a system, SB approaches toward understand-
ing bona fide PPI.
Does SB back biologists? There are specific traits that makeup PPI networks:
Everything in biology is better explained through interactions while the interactions
are a priority in accordance with the organization, cooperability, and mapping the
components in a system. The SB signifies if components interact with each other.
This led to the birth of several disciplines such as systems molecular medicine,
immunological SB, local and global metabolic profiling, systems diagnostic therapy,
1 Whither Bioinformatics? 9

and systems drug development, all budding across nascent biology disciplines.
Although the PPI are outcomes of almost all cellular processes, there is diversity
in protein interactions, that is, all proteins share common properties at a certainty.
For example, the distortion of protein interfaces leads to the development of many
diseases, and to understand its mechanism, we lead PPI experiments. When proteins
recognize specific targets and bind them, it results in conservation that depends on
structural and physicochemical properties. The nature and applications of SB with
respect to PPI were well-reviewed elsewhere (Huang 2004; Tracy 2008; Cornish-
Bowden et al. 2007; Huang and Wikswo 2006; Strömbäck et al. 2006).

1.3 Is Biology Explained Through Protein–Protein

Interaction Networks Alone?

Apart from the three most common omics-es, namely, “Gen-omics,” “Prote-omics,”
and “Transcript-omics,” bioinformatics and biology researchers have been taking up
omes and omics-es very rapidly as is evident from the use of the terms in PubMed
(Dell et al. 1996). As a result, a variety of omics disciplines such as phenomics
(Schork 1997), physiomics (Chotani et al. 2000; Gomase and Tagore 2008),
metabolomics (Kuiper et al. 2001; Fiehn 2002), lipidomics (Han and Gross 2003),
glycomics (Gronow and Brade 2001), interactomics (Govorun and Archakov 2002),
cellomics (Taylor et al. 2001) have begun to emerge, each with their own set of
instruments, techniques, reagents, and software. These have driven new areas of
research consisting of DNA and protein microarrays, mass spectrometry, and a
number of other instruments that enable high-throughput analyses.
While genomics forms a main hierarchy of classification, there are many other
omics-es that fall under a clad of primary (gen) omics’ enabled SB, for example,
functional genomics, comparative genomics, computational genomics, and
phylogenomics. With more than 1800 microbial genomes sequenced or being
sequenced today and the number still increasing, another set of omics called
metagenomics aims to access the genomic potential of an environmental sample. It
would answer some of the questions we posed in the earlier sections. This environ-
mental “omics” bridges the integration of metagenomics with complementary
approaches in microbial ecology (Schloss and Handelsman 2003).
While the mapping of PPI is a key to understand biological processes through
interactomics, many technologies have been reported to map interactions, widely
applied in yeast. At present, the number of reported yeast protein interactions truly
validated by at least one other approach is low with the amount of throughput it takes
to process (Cornell et al. 2004). This is because of the false discovery rate of proteins
interacting with their partners. With the advent of virtual interactions, the growth of
false positives also increased, thereby allowing the researchers to keep a track of
finding these false positives through statistical inference. Any dataset of interaction
map is complex while tools to decipher true positives are being developed in the
10 P. N. Suravajhala

form of markup languages such as system biology markup language (SBML)

(Hucka et al. 2004). The mapping of human–protein interaction networks is even
more complicated, suggesting that it is unreasonable to try mapping the human
interactome; instead, interaction mapping in human cell lines should be focused
along the lines of diseases or changes that can be associated with specific cells
(Figeys 2004). This “omics revolution” would force us to re-evaluate our ability to
acquire, measure, and handle large datasets. The omic platforms such as expression
arrays, MS, and other high-throughput methods have enabled quantification of
proteins and metabolites derived from complex tissues. Applying SB, the integrated
analysis of genetic, genomic, protein, metabolite, cellular, and pathway events are in
flux and interdependent. With the onset of various datasets, it necessitated the use of
a variety of analytic platforms as well as biostatistics, bioinformatics, data integra-
tion, computational biology, modeling, and knowledge assembly protocols. Such
sophisticated analyses would definitely provide new insight into the understanding
of disease processes through phenome–genome networks and interactomics studies
(Lage et al. 2007). In this regard, SB clubbed with interactomics, more appropriately
considered as a process containing a series of modules, aims to provide tools and
capabilities to carry out a wide range of tasks (Morel et al. 2004). Even as protein
analysis is known as a field of research with a long history, several developments of a
series of proteomics approaches including MS opened the door for a synergistic
combination with genomic sequence analysis, focusing on aspects of genome-wide
transcription control, regulomics. In analogy with all the other omics-es, a combi-
nation of MS-based proteomics with in silico regulomics analyses can produce
synergistic effects in the quest to understand how cells function (Werner 2004).
Carrying this further, it has been suggested that the term “translatome” could be used
to describe the members of the proteome weighted by their abundance, and the
“functome” to describe all the functions carried out by them (Greenbaum et al.
2001). However, there are still many difficulties resulting from the disorderliness
and complexity of the information. To overcome this, removing noisy data and
finding false positives could be enhanced using various tools to some degree.
However, these can also be overcome by averaging broad proteomic categories
such as those implicit in functional and structural classifications (Fig. 1.3).

1.4 Systems Biology in Wet Laboratory

Fundamental biological processes can now be studied by applying the full range of
omics technologies (genomics, transcriptomics, proteomics, metabolomics, etc.)
using the same biological sample and high-throughput methods such as MS
(McGuire et al. 2008; Kim et al. 2008). A wide array of assays including high-
throughput methods such as tandem mass spectrometry (MS/MS), yeast two hybrids
(Y2H), and pull-down assays are preferentially used to navigate them. Clearly, it
would be desirable if the concept of the sample were shared among technologies
such as MS for that, until the time a biological sample is prepared for use in a specific
1 Whither Bioinformatics? 11

Fig. 1.3 Quantitative picture of various omics and the various fields, an enthusiast can take up

omics assay, its description is inherently technology independent. However, the

compulsion for accurate analyses of all these high-throughput methods is to remove
redundant and false-positive data. Redundancy of data has been the biggest threat for
causing errors in data usage. Sharing a common informatics’ representation would
encourage data sharing, leading to a decrease in redundant data, and the potential for
error. The recent introduction of WikiProteins has been a worthy effort that brought
all annotators to come together on a common platform (Mons et al. 2008). This
would result in a significant degree of harmonization across different omics data
standardization activities, a task that is critical if we are to integrate data from these
different data sources (Morrison et al. 2006). The bioinformatics applied to omics are
varied and particularly noteworthy or characteristic of proteomics research, for
example, 2DE analysis or MS. Another important task of bioinformatics is the
prediction of functional properties through ontology-based functional networks
from a vast number of databases.
Apart from the above-discussed issues, genome technologies are being carried
out in every major model system. For example, new technologies are being devel-
oped to rapidly identify mutations or small molecules that increase the life span for
aging-related research. While the DOG recently has been known to play a role as a
model system for cancer, because of its similarities to human anatomy and physiol-
ogy, it may prove invaluable in research and development on cancer drugs (Khanna
12 P. N. Suravajhala

2006). Inversely, as dogs too naturally develop cancer they may share many
characteristics with human malignancies. This probably would accelerate genome-
wide, cross-comparison of organisms for finding the function of more genes ulti-
mately using drug discovery development.

1.4.1 Metabolomics

Metabolomics has come into sight as one of the newest “omics” science with a
dynamic portrait of the metabolic status of living systems. The analysis of the
metabolome is particularly challenging as it has it’s roots in early metabolite
profiling studies but is now a rapidly expanding area of scientific research in its
own right. It is a science employed toward the understanding of global SB (Rochfort
2005). The metabolomic tools aim to fill the gap between genotype and phenotype
permitting simultaneous monitoring molecules in a living system. The smartness of
using metabolic information could be applied in translating into diagnostic tests as
they might have the potential to impact on clinical practice and might lead to the
supplementation of traditional biomarkers of cellular integrity, cell and tissue
homeostasis, and morphological alterations that result from cell damage or death
(Claudino et al. 2007). Metabolomics has been widely applied to optimize microor-
ganisms for white biotechnology even as it spreads to the investigation of biotrans-
formation and cell culture. Together with the other more established omics
technologies, metabolomics aims to contribute to different spheres ranging from
an understanding of the in vivo function of gene products to the simulation of the
whole cell in the SB approach. This will allow the construction of designer organ-
isms and yet another science synthetic biology evolves (Oldiges et al. 2007).
Although metabolomics measures the multiparametric response of living systems
to genetic modification, there is a consistent debate of synonymy with
metabolomics. Admittedly, there is a concurrence of the former being associated
with NMR while the latter being associated with mass spectroscopy. This part of the
microbial transformation has led several standards for these two meta-omics’ deliv-
ering SB tools (Fiehn et al. 2006).

1.5 Mitochondriomics

Mitochondria are semiautonomous organelles, presumed to be the evolutionary

product of a symbiosis between a eukaryote and a prokaryote. The organelle is
present in almost all eukaryotic cells to an extent from 10^3 to 10^4 copies. The
main function of mitochondria is the production of ATP by oxidative phosphoryla-
tion and its involvement in apoptosis. The organelles contain almost exclusively
maternally inherited mtDNA, and they have specific systems for transcription,
translation, and replication of mtDNA. Mitochondrial dysfunction has been
1 Whither Bioinformatics? 13

correlated with mitochondrial diseases where the clinical pathologies are believed to
include infertility, diabetes, blindness, deafness, stroke, migraine, heart, kidney, and
liver diseases (Reichert and Neupert 2004).
Recently, cancer was added to this list when investigations into human cancer
cells from breast, bladder, neck, and lung revealed a high occurrence of mutations in
mtDNA. With the understanding of the role of mitochondria in a vast array of
pathologies, research on mitochondria and mitochondrial dysfunction has in the
last decade yielded a huge amount of data in the form of publications and databases.
Yet, the field of mitochondrial research is still far from exhaustion with many
essentials waiting to be discovered. The recent identification of a number of proteins
targeting mitochondria has enabled immense interest to understand the function of
some genes unnoticed in the mitochondrion (Calvo et al. 2006). With only 13 pro-
teins sitting inside mitochondria through oxidative phosphorylation, and more than
1500 estimated proteins targeting this tiny organelle, identifying complete protein
repertoire in this machinery could decipher the biology behind mitochondria or what
makes us breathe. A complete set of mitochondrial proteomes syntenic with other
eukaryotes has just started and there is a promise in understanding how the organelle
proteomes and interactomes could essentially be used to develop into SB (Calvo
et al. 2006).

1.6 “Omic” Challenges in Systems Biology

Bioinformatics has enabled all-against-all comparison distinguishing unique pro-

teins from proteins that are paralogs resulting from gene duplication events. The last
two decades have seen an avalanche in databases while algorithms such as BLAST
allowed such comparisons. In the post-genomic era, the genomes sequenced so far
would essentially cover the future of omics in them as they enable predicting not
only evolutionary relationships but also make use of different approaches used in
identifying the function of genes. This functional genomics is the cause of under-
standing how proteins interact with each other and network in the living organism.
The gene or protein function could be ascertained based on physiological character-
ization or if the two proteins are known to be physically interacting with each other
or virtually interacting with each other. The SB approaches in present-day bioinfor-
matics have brought in a special emphasis on association-based networks in the form
of virtual interactions, thereby making up the possibility of phenome–genome
networks grow bigger (Lage et al. 2007). Ultimately, it makes sense when such
interactions bring out a function and find a candidate for disease. The increase in
GenBank accessions resulted not only in the number of genes identified but also in
the number of citations these accessions refer to. While various databases and terms
have been defined, several omics-es are reported from time to time at http://www.
omics.org (Fig. 1.4).
The last 10 years have not only seen the rise of bioinformatics producing an
unprecedented amount of genome-scale data from many organisms but also the wet
14 P. N. Suravajhala

Fig. 1.4 Proportion of Structural Biology

research articles published Systems Biology
in different omics-es in
PubMed as on September 20.4% Next generation
1, 2012 sequencing
Functional
41.1% genomics
4%

34.6%

laboratory research community has been successful in exploring these data on using
bioinformatics many challenges still persist. One of them is the effective integration
of datasets directly into approaches based on mathematical modeling of biological
systems. This is where SB has bud resulting in top–down and bottom–up
approaches. The advent of functional genomics has enabled the molecular biosci-
ences to come a long way toward characterizing the molecular constituents of life.
Yet, the challenge for biology overall is to understand how organisms’ function. By
discovering how function arises in dynamic interactions, SB is everywhere
addressing the missing links between molecules and physiology. Top–down SB
identifies molecular interaction networks on the basis of correlated molecular behav-
ior observed in genome-wide “omics” studies. On the other hand, bottom–up SB
examines the mechanisms through which functional properties arise in the interac-
tions of known components. Applications in cancer are a good example to counteract
these two major types of complementary strategies (Stransky et al. 2007). Several
web-based repositories have been established to store protein and peptide identifi-
cations derived from MS data, and a similar number of peptide identification
software pipelines and workflows have emerged to deliver identifications to these
repositories. Integrated data analysis is introduced as the intermediate level of an SB
approach and as a supplementary to bioinformatics to analyze different “omics”
datasets, that is, genome-wide measurements of transcripts, protein levels or PPI,
and metabolite levels aiming at generating a coherent understanding of biological
function (Steinfath et al. 2007). Furthermore, existing and potential problems/solu-
tions such as de facto experimental and the following bioinformatics challenges
might hold prospective in the near future:
1. Challenges in high-dimensional biology (HDB): Recently, the term HDB has
been proposed for investigations involving high-throughput data (Mehta et al.
2006). The HDB includes whole-genome sequences, expression levels of genes,
protein abundance measurements, and other permutations. The identification of
biomarkers, the effects of mutations and drug treatments, and the investigation of
1 Whither Bioinformatics? 15

diseases as multifactor phenomena can now be accomplished on an unprece-

dented scale.
2. Finding the function of HP: Another feature of PPI map is to find the function of
unknown proteins. PPI has become a very common step in the annotation of a
protein. Various tools such as iHOP (http://www.ihop-net.org), STRING (http://
string.embl.de), GeneMania (http://www.genemania.org), and so on, aid the
researchers to find if there are interacting partners of protein of interest. The
data could be visualized through tools such as Cytoscape (www.cytoscape.org),
VisANt (http://www.visant.bu.edu), and Osprey (http://biodata.mshri.on.ca/
osprey/servlet/Index), and so on, for further analyses. The nearest partners
would essentially mean that the hypothetical or uncharacterized protein could
play a function similar to its interactor(s). In the context of PPI networks, we
could consider if a model is to be developed from the network or a network is to
be generated with an already established model. Precisely, the putative function
of a protein could be better known from a PPI network to develop a model from
it. Information on “known” or “unknown” PPI is still mostly limited but inte-
grating tools such as these could generalize a way to find bona fide function.

1.7 Are Interactions Based on the Nature of Binding?

Does close homology between two proteins confer that they do interact in the same
manner? Yes, they do and confer evolutionary constraints in lieu of structural
divergence while remotely related proteins have a different interaction mode
(Drummond et al. 2005). Also, conservation of protein interface indicates the
average conservation of the rest of the protein. While all these forms an integral
part of SB, apart from the novel interactions that arise based on the type of
homology, there are interactions based on the binding entity, namely, stable and
transient. The former interactions are consistent and bookmarked while the latter is
temporary. There are interacting proteins that might co-express indicating that the
expressed proteins, which evolve slowly are normalized wherein the normalized
difference between the absolute expression data is calculated based on several tools
such as microarrays (Drummond et al. 2005). However, there are other techniques
such as density gradient and virtual pull-down assay methods cited as above
beginning to be understood and substantiate above views.
As thousands of new genes are identified in genomics efforts, the rush is on to
learn something about the functional roles of the proteins encoded by those genes.
Clues to protein functions, activation states, and PPI have been revealed in focused
studies of protein localization. A meta-analysis of data derived from genome-wide
studies of aging in simple eukaryotes will allow the identification of conserved
determinants of longevity that can be tested in other mammals (Khanna 2006;
Kaeberlein 2004). Adding to the various high-throughput methods, technical break-
throughs such as GFP protein tagging and recombinase clones, large-scale screens of
16 P. N. Suravajhala

protein localization are now being undertaken to understand the function of the
proteins (O’Rourke et al. 2005).

1.8 Fundamental and Best Practiced Tools for Annotating

Proteins and Genes

In the recent past, various bioinformatics tools have been developed that allow
researchers to compare genomic and proteomic repertoire. Comparative studies
using algorithms such as Blast and databases are carried out to distinguish unique
proteins from paralogs, which later might have resulted from gene duplication
events. The genomes sequenced so far were helpful in predicting not only evolu-
tionary relationships but also identified function for the genes through functional
genomics. Although many methods are being employed by researchers, screening of
proteins for novel translatable candidates is not often used and the researcher
repeatedly performs the screening with laborious wet laboratory experiments. To
increase the sensitivity, further clues on tissues and development stages from the
queried gene’s sequences could be surveyed using tools such as gene expression
omnibus (GEO) or UniGene-EST or cDNA profile database. Furthermore, protein
link to the genomic location specified by transcript mapping, radiation hybrid
mapping, genetic mapping, or cytogenetic mapping as available from GenBank
resources would improve the understanding of protein annotation. Besides this,
whether or not a protein contains a polyadenylation signal could be an added
knowledge to meet the criteria of well-annotated proteins. This is because tools
such as MEME reveal many 30 UTRs forming conserved motifs, which indicates
these regions appear more conserved than expected. This means, higher the conser-
vation, greater the duplications and greater is the chance of being not annotated or
“hypothetical.” There seem to be many unique genes that are overrepresented in the
form of duplications; a simple search in GenBank gene list would reveal that there
are several accessions duplicated. For example, in the case of the gene FusA2a, bona
fide accession is mapped to CAD92986, and yet, a few of the isoforms/unique genes
remain unknown (e.g., CAD93127). In summary, there could be many proteins less
annotated, and yet many tools are known to describe the function. This leaves to beg
a question, what would be the fate of proteins that cannot be annotated through some
tools, or in contrast how many best tools are used to describe or annotate a protein?
Apart from BLAST and FASTA, the sequence-based feature annotation is
applied by RefSeq using several tools, namely, BEAUTY X-Blast Enhanced Align-
ment Utility, and PROSITE. While many other variants of BLAST including PSI
Blast and PHI Blast, sequence alignments using ClustalW, ClustalX, and Cobalt are
used, not all the tools are used in tandem to eliminate false positives. Whether the
protein is soluble or insoluble is known through TopPred; the topology of protein
with the orientation and location of transmembrane helices attribute to the function.
Additionally, orthology mapping using tools such as HomoMINT are used, which
1 Whither Bioinformatics? 17

increases the chance of the protein annotation. With the central dogma beyond the
age today in bioinformatics, namely, sequence specifies structure and function;
annotations have become mightier to further manually curate allowing researchers
to perform experimental analyses for some proteins. The structures of proteins not
only provide functions but the shapes exhibited by the proteins allow them to interact
selectively with other proteins or molecules. This specificity is the key for the
proteins to interact with another protein, thereby inferring the function. However,
most of the bioinformatics analyses are misleading unless biochemical characteri-
zation is carried out. Furthermore, the protein annotation has gained much impor-
tance with the introduction of many metazoan genome sequencing projects in
addition to the 1000 genomes project that is in progress. With 40–50% of identified
genes corresponding to proteins of unknown function, the functional structural
annotation screening technology using NMR (FAST-NMR) has been developed to
assign a biological function which is based on the principle that a biological function
can be described based on the basic dogma of biochemistry that the proteins with
similar functions will have similar active sites and exhibit similar ligand-binding
interactions, although there is a global difference in sequence and structure. Tools
such as combinatorial extension which confer structure similarity, DALI for NMR,
finally determining function, PvSOAR, and Profunc—given a 3D structure, aims at
identifying a protein’s function has been widely used. However, there are many
other methods such as the Rosetta Stone method, phylogenetic profiling method, and
conserved gene neighbors that have been widely employed and being accepted by
the scientific community.
Biological function of proteins would help in the identification of novel drug
targets and helps reduce the extensive cost of practical examinations on several
candidates. With the enormous amount of sequence and structure information
availability, innumerable automated annotation tools for proteins have also been
generated. One such example is the automated protein annotation tool (APAT),
which uses a markup language concept to provide wrappers for several kinds of
protein annotations. While FFPred is available to predict molecular function for
orphan and unannotated protein sequences, the method has been optimized for
performance using a protein feature-based method through support vector machines
(SVMs) that does not require prior identification of protein sequence homologs. It
works on the premise of posttranslational modifications, Gene Ontology, and local-
ization features of proteins. Yet another tool, namely, VICMpred, aids in broad
functional classification of proteins of bacteria into virulence factors, information
molecule, cellular process, and metabolism molecule. The VICMpred server uses an
SVM-based method having patterns, amino acid, and dipeptide composition of
bacterial protein sequences. ConSeq and ConSurf have been widely applied in
predicting functional/structural sites in a protein using conservation and
hypervariation.
The final part of annotation can be studied through interactions and associations.
All interactions are associations, while not all associations are interactions. The
association tools, namely, search tool for the retrieval of interacting genes/proteins
(STRING), GeneCards, IntAct, MINT, biomolecular interaction network database
Another Random Scribd Document
with Unrelated Content
indulging in any explanations, and obviously with great moral effort,
Willett staggered dizzily down to the cellar and tried the fateful
platform before the tubs. It was unyielding. Crossing to where he had
left his yet-unused tool satchel the day before, he obtained a chisel
and began to pry up the stubborn planks one by one. Underneath the
smooth concrete was still visible, but of any opening or perforation
there was no longer a trace. Nothing yawned this time to sicken the
mystified father who had followed the doctor downstairs; only the
smooth concrete underneath the planks—no noisome well, no world
of subterrene horrors, no secret library, no Curwen papers, no
nightmare pits of stench and howling, no laboratory or shelves or
chiseled formulae, no—Dr. Willett turned pale, and clutched at the
younger man. "Yesterday," he asked softly, "did you see it here—and
smell it?" And when Mr. Ward, himself transfixed with dread and
wonder, found strength to nod an affirmative, the physician gave a
sound half a sigh and half a gasp, and nodded in turn. "Then I will
tell you," he said.
So for an hour, in the sunniest room they could find upstairs, the
physician whispered his frightful tale to the wondering father. There
was nothing to relate beyond the looming up of that form when the
greenish-black vapor from the kylix parted, and Willett was too tired
to ask himself what had really occurred. There were futile, bewildered
head-shakings from both men, and once Mr. Ward ventured a hushed
suggestion, "Do you suppose it would be of any use to dig?" The
doctor was silent, for it seemed hardly fitting for any human brain to
answer when powers of unknown spheres had so vitally encroached
on this side of the Great Abyss. Again Mr. Ward asked, "But where did
it go? It brought you here, you know, and it sealed up the hole
somehow."
And Willett again let silence answer for him.
But after all, this was not the final phase of the matter. Reaching for
his handkerchief before rising to leave, Dr. Willett's fingers closed
upon a piece of paper in his pocket which had not been there before,
and which was companioned by the candles and matches he had
seized in the vanished vault. It was a common sheet, torn obviously
from the cheap pad in that fabulous room of horror somewhere
underground, and the writing upon it was that of an ordinary lead
pencil—doubtless the one which had lain beside the pad. It was
folded very carelessly, and beyond the faint acrid scent of the cryptic
chamber bore no print or mark of any world but this. But in the text
itself it did indeed reek with wonder; for here was no script of any
wholesome age, but the labored strokes of mediaeval darkness,
scarcely legible to the laymen who now strained over it, yet having
combinations of symbols which seemed vaguely familiar. The briefly
scrawled message was this, and its mystery lent purpose to the
shaken pair, who forthwith walked steadily out to the Ward car and
gave orders to be driven first to a quiet dining place and then to the
John Hay Library on the hill.

At the library it was easy to find good manuals of palaeography, and

over these the two men puzzled till the lights of evening shone out
from the great chandelier. In the end they found what was needed.
The letters were indeed no fantastic invention, but the normal script
of a very dark period. They were the pointed Saxon minuscules of the
eighth or ninth century A. D., and brought with them memories of an
uncouth time when under a fresh Christian veneer ancient faiths and
ancient rites stirred stealthily, and the pale moon of Britain looked
sometimes on strange deeds in the Roman ruins at Caerleon and
Hexhaus, and by the Towers along Hadrian's crumbling wall. The
words were in such Latin as a barbarous age might remember
—"Corvinus, necandus est. Cadaver aq(ua) forti dissolvendum, nec
aliq(ui)d retinendum. Tace ut potes."—which may roughly be
translated, "Curwen must be killed. The body must be dissolved in
aqua fortis, nor must anything be retained. Keep silence as best you
are able."
Willett and Mr. Ward were mute and baffled. They had met the
unknown, and found that they lacked emotions to respond to it as
they vaguely believed they ought. With Willett, especially, the
capacity for receiving fresh impressions of awe was well-nigh
exhausted; and both men sat still and helpless till the closing of the
library forced them to leave. Then they drove listlessly to the Ward
mansion in Prospect Street, and talked to no purpose into the night.
The doctor rested toward morning, but did not go home. And he was
still there Sunday noon when a telephone message came from the
detectives who had been assigned to look up Dr. Allen.
Mr. Ward, who was pacing nervously about in a dressing-gown,
answered the call in person; and told the men to come up early the
next day when he heard their report was almost ready. Both Willett
and he were glad that this phase of the matter was taking form, for
whatever the origin of the strange minuscule message, it seemed
certain that the "Curwen" who must be destroyed could be no other
than the bearded and spectacled stranger. Charles had feared this
man, and had said in the frantic note that he must be killed and
dissolved in acid. Allen, moreover, had been receiving letters from the
strange wizards in Europe under the name of Curwen, and palpably
regarded himself as an avatar of the bygone necromancer. And now
from a fresh and unknown source had come a message saying that
"Curwen" must be killed and dissolved in acid. The linkage was too
unmistakable to be factitious; and besides, was not Allen planning to
murder young Ward upon the advice of the creature called
Hutchinson? Of course, the letter they had seen had never reached
the bearded stranger; but from its text they could see that Allen had
already formed plans for dealing with the youth if he grew too
"squeamish." Without doubt, Allen must be apprehended; and even if
the most drastic directions were not carried out, he must be placed
where he could inflict no harm upon Charles Ward.
That afternoon, hoping against hope to extract some gleam of
information anent the inmost mysteries from the only available one
capable of giving it, the father and the doctor went down the bay and
called on young Charles at the hospital. Simply and gravely Willett
told him all he had found, and noticed how pale he turned as each
description made certain the truth of the discovery. The physician
employed as much dramatic effect as he could, and watched for a
wincing on Charles' part when he approached the matter of the
covered pits and the nameless hybrids within. But Ward did not
wince. Willett paused, and his voice grew indignant as he spoke of
how the things were starving. He taxed the youth with shocking
inhumanity, and shivered when only a sardonic laugh came in reply.
For Charles, having dropped as useless his pretense that the crypt did
not exist, seemed to see some ghastly jest in this affair; and chuckled
hoarsely at something which amused him. Then he whispered, in
accents doubly terrible because of the cracked voice he used, "Damn
'em, they do eat, but they don't need to! That's the rare part! A
month, you say, without food? Lud, Sir, you be modest! D'ye know,
that was the joke on poor old Whipple with his virtuous bluster! Kill
everything off, would he? Why, damme, he was half-deaf with the
noise from Outside and never saw or heard aught from the wells. He
never dreamed they were there at all! Devil take ye, those cursed
things have been howling down there ever since Curwen was done
for a hundred and fifty-seven years gone!"
But no more than this could Willett get from the youth. Horrified, yet
almost convinced against his will, he went on with his tale in the
hope that some incident might startle his auditor out of the mad
composure he maintained. Looking at the youth's face, the doctor
could not but feel a kind of terror at the changes which recent
months had wrought. Truly, the boy had drawn down nameless
horrors from the skies. When the room with the formulae and the
greenish dust was mentioned, Charles shewed his first sign of
animation. A quizzical look overspread his face as he heard what
Willett had read on the pad, and he ventured the mild statement that
those notes were old ones, of no possible significance to anyone not
deeply initiated in the history of magic. "But," he added, "had you but
known the words to bring up that which I had out in the cup, you
had not been here to tell me this. 'Twas Number 118, and I conceive
you would have shook had you looked it up in my list in t'other room.
'Twas never raised by me, but I meant to have it up that day you
came to invite me hither."
Then Willett told of the formula he had spoken and of the greenish-
black smoke which had arisen; and as he did so he saw true fear
dawn for the first time on Charles Ward's face. "It came, and you be
here alive!" As Ward croaked the words his voice seemed almost to
burst free of its trammels and sink to cavernous abysses of uncanny
resonance. Willett, gifted with a flash of inspiration, believed he saw
the situation, and wove into his reply a caution from a letter he
remembered. "No. 118, you say? But don't forget that stones are all
changed now in nine grounds out of ten. You are never sure till you
question!" And then, without warning, he drew forth the minuscule
message and flashed it before the patient's eyes. He could have
wished no stronger result, for Charles Ward fainted forthwith.

All this conversation, of course, had been conducted with the

greatest secrecy lest the resident alienists accuse the father and the
physician of encouraging a madman in his delusions. Unaided, too,
Dr. Willett and Mr. Ward picked up the stricken youth and placed him
on the couch. In reviving, the patient mumbled many times of some
word which he must get to Orne and Hutchinson at once; so when
his consciousness seemed fully back the doctor told him that of those
strange creatures at least one was his bitter enemy, and had given
Dr. Allen advice for his assassination. This revelation produced no
visible effect, and before it was made the visitors could see that their
host had already the look of a hunted man. After that he would
converse no more, so Willett and the father departed presently;
leaving behind a caution against the bearded Allen, to which the
youth only replied that this individual was very safely taken care of,
and could do no one any harm even if he wished.
This was said with an almost evil chuckle very painful to hear. They
did not worry about any communications Charles might write to that
monstrous pair in Europe, since they knew that the hospital
authorities seized all outgoing mail for censorship and would pass no
wild or outré-looking missive.
There is, however, a curious sequel to the matter of Orne and
Hutchinson, if such indeed the exiled wizards were. Moved by some
vague presentiment amidst the horrors of that period, Willett
arranged with an international press-cutting bureau for accounts of
notable current crimes and accidents in Prague and in eastern
Transylvania; and after six months believed that he had found two
very significant things amongst the multifarious items he received
and had translated. One was the total wrecking of a house by night
in the oldest quarter of Prague, and the disappearance of the evil old
man called Josef Nadeh, who had dwelt in it alone ever since anyone
could remember. The other was a titan explosion in the Transylvania
mountains east of Rakus, and the utter extirpation with all its inmates
of the ill-regarded Castle Ferenczy, whose master was so badly
spoken of by peasants and soldiery alike that he would shortly have
been summoned to Bucharest for serious questioning had not this
incident cut off a career already so long as to antedate all common
memory. Willett maintains that the hand which wrote those
minuscules was able to wield stronger weapons as well; and that
while Curwen was left to him to dispose of, the writer felt able to find
and deal with Orne and Hutchinson itself. Of what their fate may
have been the doctor strives sedulously not to think.
The following morning Dr. Willett hastened to the Ward home to be
present when the detectives arrived. Allen's destruction or
imprisonment—or Curwen's, if one might regard the tacit claim to
reincarnation as valid—he felt must be accomplished at any cost, and
he communicated this conviction to Mr. Ward as they sat waiting for
the men to come. They were downstairs this time, for the upper
parts of the house were beginning to be shunned because of a
peculiar nauseousness which hung indefinitely about; a nauseousness
which the older servants connected with some curse left by the
vanished Curwen portrait.
At nine o'clock the three detectives presented themselves and
immediately delivered all that they had to say. They had not,
regrettably enough, located the Brava Tony Gomes as they had
wished, nor had they found the least trace of Dr. Allen's source or
present whereabouts; but they had managed to unearth a
considerable number of local impressions and facts concerning the
reticent stranger. Allen had struck Pawtuxet people as a vaguely
unnatural being and there was an universal belief that his thick
Vandyke beard was either dyed or false—a belief conclusively upheld
by the finding of such a false beard, together with a heavy pair of
dark glasses, in his room at the fateful bungalow. His voice, Mr. Ward
could well testify from his one telephone conversation, had a depth
and hollowness that could not be forgotten; and his glance seemed
malign even through his smoked and horn-rimmed glasses. One
shopkeeper, in the course of negotiations, had seen a specimen of his
handwriting and declared it was very queer and crabbed; this being
confirmed by penciled notes of no clear meaning found in his room
and identified by the merchant.
In connection with the vampirism ructions of the preceding summer,
a majority of the gossips believed that Allen rather than Ward was
the actual vampire. Statements were also obtained from the officials
who had visited the bungalow after the unpleasant incident of the
motor truck robbery. They had felt less of the sinister in Dr. Allen, but
had recognized him as the dominant figure in the queer shadowy
cottage. The place had been too dark for them to observe him
clearly, but they would know him again if they saw him. His beard
had looked odd, and they thought he had some slight scar above his
dark spectacled right eye. As for the search of Allen's room, it yielded
nothing definite save the beard and glasses, and several penciled
notes in a crabbed writing, which Willett at once saw was identical
with that shared by the old Curwen manuscripts and by the
voluminous recent notes of young Ward found in the vanished
catacombs of horror.
Dr. Willett and Mr. Ward caught something of a profound, subtle, and
insidious cosmic fear from this data as it was gradually unfolded, and
almost trembled in following up the vague, mad thought which had
simultaneously reached their minds. The false beard and glasses, the
crabbed Curwen penmanship—the old portrait and its tiny scar—and
the altered youth in the hospital with such a scar—that deep, hollow
voice on the telephone—was it not of this that Mr. Ward was
reminded when his son barked forth those pitiable tones to which he
now claimed to be reduced? Who had ever seen Charles and Allen
together? Yes, some officials had once, but who later on? Was it not
when Allen left that Charles suddenly lost his growing fright and
began to live wholly at the bungalow? Curwen—Allen—Ward—in what
blasphemous and abominable fusion had two ages and two persons
become involved? That damnable resemblance of the picture to
Charles—had it not used to stare and stare, and follow the boy
around the room with its eyes? Why, too, did both Allen and Charles
copy Joseph Curwen's handwriting, even when alone and off guard?
And then the frightful work of those people—the lost crypt of horrors
that had aged the doctor overnight; the starved monsters in the
noisome pits; the awful formula which had yielded such nameless
results; the message in minuscules found in Willett's pocket; the
papers and the letters and all the talk of graves and "salts" and
discoveries—whither did everything lead? In the end Mr. Ward did the
most sensible thing. Steeling himself against any realization of why
he did it, he gave the detectives an article to be shewn to such
Pawtuxet shopkeepers as had seen the portentous Dr. Allen. That
article was a photograph of his luckless son, on which he now
carefully drew in ink the pair of heavy glasses and the black pointed
beard, which the men had brought from Allen's room.
For two hours he waited with the doctor in the oppressive house
where fear and miasma were slowly gathering as the empty panel in
the upstairs library leered and leered and leered. Then the men
returned. Yes, the altered photograph was a very passable likeness of
Dr. Allen. Mr. Ward turned pale, and Willett wiped a suddenly
dampened brow with his handkerchief. Allen—Ward—Curwen—it was
becoming too hideous for coherent thought. What had the boy called
out of the void, and what had it done to him? What really had
happened from first to last? Who was this Allen who sought to kill
Charles as too "squeamish," and why had his destined victim said in
the postscript to that frantic letter that he must be so completely
obliterated in acid? Why, too, had the minuscule message, of whose
origin no one dared think, said that "Curwen" must be likewise
obliterated? What was the change, and when had the final stage
occurred? That day when his frantic note was received—he had been
nervous all the morning, then there was an alteration. He had slipped
out unseen and swaggered boldly in past the men hired to guard
him. That was the time, when he was out. But no—had he not cried
out in terror as he entered his study—this very room? What had he
found there? Or wait—what had found him? That simulacrum which
brushed boldly in without having been seen to go—was that an alien
shadow and a horror forcing itself upon a trembling figure which had
never gone out at all? Had not the butler spoken of queer noises?

Willett rang for the man and asked him some low-toned questions. It
had, surely enough, been a bad business. There had been noises—a
cry, a gasp, a choking, and a sort of clattering or creaking or
thumping, or all of these. And Mr. Charles was not the same when he
stalked out without a word. The butler shivered as he spoke, and
sniffed at the heavy air that blew down from some open window
upstairs. Terror had settled definitely upon the house, and only the
businesslike detectives failed to imbibe a full measure of it. Even they
were restless, for this case had held vague elements in the
background which pleased them not at all. Dr. Willett was thinking
deeply and rapidly, and his thoughts were terrible ones. Now and
then he would almost break into muttering as he ran over in his head
a new, appalling, and increasingly conclusive chain of nightmare
happenings.
Then Mr. Ward made a sign that the conference was over, and
everyone save him and the doctor left the room. It was noon now,
but shadows as of coming night seemed to engulf the phantom-
haunted mansion. Willett began talking very seriously to his host, and
urged that he leave a great deal of the future investigation to him.
There would be, he predicted, certain obnoxious elements which a
friend could bear better than a relative. As family physician he must
have a free hand, and the first thing he required was a period alone
and undisturbed in the abandoned library upstairs, where the ancient
overmantel had gathered about itself an aura of noisome horror more
intense than when Joseph Curwen's features themselves glanced
slyly down from the painted panel.
Mr. Ward, dazed by the flood of grotesque morbidities and
unthinkably maddening suggestions that poured in upon him from
every side, could only acquiesce; and half an hour later the doctor
was locked in the shunned room with the paneling from Olney Court.
The father, listening outside, heard fumbling sounds of moving and
rummaging as the moments passed; and finally a wrench and a
creak, as if a tight cupboard door were being opened. Then there
was a muffled cry, a kind of snorting choke, and a hasty slamming of
whatever had been opened. Almost at once the key rattled and
Willett appeared in the hall, haggard and ghastly, and demanding
wood for the real fireplace on the south wall of the room. The
furnace was not enough, he said; and the electric log had little
practical use. Longing yet not daring to ask questions, Mr. Ward gave
the requisite orders and a man brought some stout pine logs,
shuddering as he entered the tainted air of the library to place them
in the grate. Willett meanwhile had gone up to the dismantled
laboratory and brought down a few odds and ends not included in
the moving of the July before. They were in a covered basket, and
Mr. Ward never saw what they were.
Then the doctor locked himself up in the library once more, and by
the clouds of smoke which rolled down past the windows from the
chimney it was known that he had lighted the fire. Later, after a great
rustling of newspapers, that odd wrench and creaking were heard
again; followed by a thumping which none of the eavesdroppers
liked. Thereafter two suppressed cries of Willett's were heard, and
hard upon these came a swishing rustle of indefinable hatefulness.
Finally the smoke that the wind beat down from the chimney grew
very dark and acrid, and everyone wished that the weather had
spared them this choking and venomous inundation of peculiar
fumes. Mr. Ward's head reeled, and the servants all clustered
together in a knot to watch the horrible black smoke swoop down.
After an age of waiting the vapors seemed to lighten, and half-
formless sounds of scraping, sweeping, and other minor operations
were heard behind the bolted door. And at last, after the slamming of
some cupboard within, Willett made his appearance, sad, pale and
haggard, and bearing the cloth-draped basket he had taken from the
upstairs laboratory. He had left the window open, and into that once
accursed room was pouring a wealth of pure, wholesome air to mix
with a queer new smell of disinfectants. The ancient overmantel still
lingered; but it seemed robbed of malignity now, and rose as calm
and stately in its white paneling as if it had never borne the picture of
Joseph Curwen. Night was coming on, yet this time its shadows held
no latent fright, but only a gentle melancholy. Of what he had done
the doctor would never speak. To Mr. Ward he said, "I can answer no
questions, but I will say that there are different kinds of magic. I
have made a great purgation. Those in this house will sleep the
better for it."
That Dr. Willett's "purgation" had been an ordeal almost as nerve-
racking in its way as his hideous wandering in the vanished crypt is
shewn by the fact that the elderly physician gave out completely as
soon as he reached home that evening. For three days he rested
constantly in his room, though servants later muttered something
about having heard him after midnight on Wednesday, when the
outer door softly opened, and closed with phenomenal softness.
Servants' imaginations, fortunately, are limited, else comment might
have been excited by an item in Thursday's Evening Bulletin which
ran as follows:

North End Ghouls Again Active

After a lull of ten months since the dastardly vandalism in the
Weeden lot at the North Burial Ground, a nocturnal prowler was
glimpsed early this morning in the same cemetery by Robert Hart,
the night watchman. Happening to glance for a moment from his
shelter at about two a.m., Hart observed a glow of a lantern or
pocket torch not far to the northward, and upon opening the door
detected the figure of a man with a trowel very plainly silhouetted
against a nearby electric light. At once starting in pursuit, he saw the
figure dart hurriedly toward the main entrance, gaining the street and
losing himself among the shadows before approach or capture was
possible.
Like the first of the ghouls active during the past year, this intruder
had done no real damage before detection. A vacant part of the Ward
lot shewed signs of a little superficial digging, but nothing even
nearly the size of a grave had been attempted, and no previous grave
had been disturbed.
Hart, who cannot describe the prowler except as a small man
probably having a full beard, inclines to the view that all three of the
digging incidents have a common source; but police from the Second
Station think otherwise on account of the savage nature of the
second incident, where an ancient coffin was removed and its
headstone violently shattered.
The first of the incidents, in which it is thought an attempt to bury
something was frustrated, occurred a year ago last March, and has
been attributed to bootleggers seeking a cache. It is possible, says
Sergeant Riley, that this third affair is of similar nature. Officers at the
Second Station are taking especial pains to capture the gang of
miscreants responsible for these repeated outrages.
All day Thursday Dr. Willett rested as if recuperating from something
past or nerving himself for something to come. In the evening he
wrote a note to Mr. Ward, which was delivered the next morning and
which caused the half-dazed parent to ponder long and deeply. Mr.
Ward had not been able to go down to business since the shock of
Monday with its baffling reports and its sinister "purgation," but he
found something calming about the doctor's letter in spite of the
despair it seemed to promise and the fresh mysteries it seemed to
evoke.

10 Barnes St.,
Providence, R. I.,
April 12, 1928.
Dear Theodore:
I feel that I must say a word to you before doing what I am going to
do tomorrow. It will conclude the terrible business we have been
going through (for I feel that no spade is ever likely to reach that
monstrous place we know of), but I'm afraid it won't set your mind at
rest unless I expressly assure you how very conclusive it is.
You have known me ever since you were a small boy, so I think you
will not distrust me when I hint that some matters are best left
undecided and unexplored. It is better that you attempt no further
speculation as to Charles's case, and almost imperative that you tell
his mother nothing more than she already suspects. When I call on
you tomorrow Charles will have escaped. That is all which need
remain in anyone's mind. He was mad, and he escaped.
So don't ask me any questions when I call. It may be that something
will go wrong, but I'll tell you if it does. I don't think it will. There will
be nothing more to worry about, for Charles will be very, very safe.
He is now—safer than you dream. You need hold no fears about
Allen, and who or what he is. He forms as much a part of the past as
Joseph Curwen's picture, and when I ring your doorbell you may feel
certain that there is no such person. And what wrote that minuscule
message will never trouble you or yours.
But you must steel yourself to melancholy, and prepare your wife to
do the same. I must tell you frankly that Charles's escape will not
mean his restoration to you. He has been afflicted with a peculiar
disease, as you must realize from the subtle physical as well as
mental changes in him, and you must not hope to see him again. He
stumbled on things no mortal ought ever to know, and reached back
through the years as no one ever should reach; and something came
out of those years to engulf him.
And now comes the matter in which I must ask you to trust me most
of all. For there will be, indeed, no uncertainty about Charles's fate.
In about a year, say, you can if you wish devise a suitable account of
the end, for the boy will be no more. You can put up a stone in your
lot at the North Burial ground exactly ten feet west of your father's
and facing the same way, and that will mark the true resting-place of
your son. Nor need you fear that it will mark any abnormality or
changeling. The ashes in that grave will be those of your own
unaltered bone and sinew—of the real Charles Dexter Ward whose
mind you watched from infancy—the real Charles with the olive-mark
on his hip and without the black witch-mark on his chest or the pit on
his forehead. The Charles who never did actual evil, and who will
have paid with his life for his "squeamishness."
That is all. Charles will have escaped, and a year from now you can
put up his stone. Do not question me tomorrow. And believe that the
honour of your ancient family remains untainted now, as it has been
at all times in the past.
With profoundest sympathy, and exhortations to fortitude, calmness,
and resignation, I am ever
Sincerely your friend,
 Marinus B. Willett.
So on the morning of Friday, April 13, 1928, Marinus Bicknell Willett
visited the room of Charles Dexter Ward at Dr. Waite's private
hospital on Conanicut Island. After the interchange of a few strained
formalities, a new element of constraint crept in, as Ward seemed to
read behind the doctor's masklike face a terrible purpose which had
never been there before.
Ward actually turned pale, and the doctor was the first to speak.
"More," he said, "has been found out, and I must warn you fairly that
a reckoning is due."
"Digging again, and coming upon more poor starving pets?" was the
ironic reply. It was evident that the youth meant to shew bravado to
the last.
"No," Willett slowly rejoined, "this time I did not have to dig. We have
had men looking up Dr. Allen, and they found the false beard and
spectacles in the bungalow!"
"Excellent," commented the disquieted host in an effort to be wittily
insulting, "and I trust they proved more becoming than the beard and
glasses you now have on!"
"They would become you very well," came the even and studied
response, "as indeed they seem to have done."
As Willett said this, it almost seemed as though a cloud passed over
the sun; though there was no change in the shadows on the floor.
Then Ward ventured:
"And is this what asks so hotly for a reckoning? Suppose a man does
find it now and then useful to be twofold?"
"No," said Willett gravely, "again you are wrong. It is no business of
mine if any man seeks duality; provided he has any right to exist at
all, and provided he does not destroy what called him out of space."
Ward now started violently. "Well, Sir, what have ye found, and what
d'ye want with me?"
The doctor let a little time elapse before replying, as if choosing his
words for an effective answer.
"I have found," he finally intoned, "something in a cupboard behind
an ancient overmantel where a picture once was, and I have burned
it and buried the ashes where the grave of Charles Dexter Ward
ought to be."
The madman choked and sprang from the chair in which he had been
sitting:
"Damn ye, who did ye tell—and who'll believe it was he after these
full two months, with me alive? What d'ye mean to do?"
Willett, though a small man, actually took on a kind of judicial
majesty as he calmed the patient with a gesture.
"I have told no one. This is no common case—it is a madness out of
time and a horror from beyond the spheres which no police or
lawyers or courts or alienists could ever fathom or grapple with. You
cannot deceive me, Joseph Curwen, for I know that your accursed
magic is true!
"I know how you wove the spell that brooded outside the years and
fastened on your double and descendant; I know how you drew him
into the past and got him to raise you up from your detestable grave;
I know how he kept you hidden in his laboratory while you studied
modern things and roved abroad as a vampire by night, and how you
later shewed yourself in beard and glasses that no one might wonder
at your godless likeness to him; I know what you resolved to do
when he balked at your monstrous rifling of the world's tombs, and at
what you planned afterward, and I know how you did it.
"You left off your beard and glasses and fooled the guards around the
house. They thought it was he who went in, and they thought it was
he who came out when you had strangled and hidden him. But you
hadn't reckoned on the different contacts of two minds. You were a
fool, Curwen, to fancy that a mere visual identity would be enough.
Why didn't you think of the speech and the voice and the
handwriting? It hasn't worked, you see, after all. You know better
than I who or what wrote that message in minuscules, but I will warn
you it was not written in vain. There are abominations and
blasphemies which must be stamped out, and I believe that the
writer of those words will attend to Orne and Hutchinson. One of
those creatures wrote you once, 'do not call up any that you cannot
put down.' Curwen, a man can't tamper with Nature beyond certain
limits, and every horror you have woven will rise up to wipe you out."

But here the doctor was cut short by a convulsive cry from the
creature before him. Hopelessly at bay, weaponless, and knowing
that any show of physical violence would bring a score of attendants
to the doctor's rescue, Joseph Curwen had recourse to his one
ancient ally, and began a series of cabalistic motions with his
forefingers as his deep, hollow voice, now unconcealed by feigned
hoarseness, bellowed out the opening words of a terrible formula.
"PER ADONAI ELOIM, ADONAI JEHOVA, ADONAI SABAOTH,
METRATON...."
But Willett was too quick for him. Even as the dogs in the yard
outside began to howl, and even as a chill wind sprang suddenly up
from the bay, the doctor commenced the solemn and measured
intonation of that which he had meant all along to recite. An eye for
an eye—magic for magic—let the outcome shew how well the lesson
of the abyss had been learned! So in a clear voice Marinus Bicknell
Willett began the second of that pair of formulae whose first had
raised the writer of those minuscules—the cryptic invocation whose
heading was the Dragon's Tail, sign of the descending node—
At the very first word from Willett's mouth the previously commenced
formula of the patient stopped short. Unable to speak, the monster
made wild motions with his arms until they too were arrested. When
the awful name of Yog-Sothoth was uttered, the hideous change
began. It was not merely a dissolution, but rather a transformation or
recapitulation; and Willett shut his eyes lest he faint before the rest
of the incantation could be pronounced.
But he did not faint, and that man of unholy centuries and forbidden
secrets never troubled the world again. The madness out of time had
subsided, and the case of Charles Dexter Ward was closed. Opening
his eyes before staggering out of that room of horror, Dr. Willett saw
that what he had kept in memory had not been kept amiss. There
had, as he had predicted, been no need for acids. For like his
accursed picture a year before, Joseph Curwen now lay scattered on
the floor as a thin coating of fine bluish-gray dust.
 *** END OF THE PROJECT GUTENBERG EBOOK THE CASE OF
CHARLES DEXTER WARD ***

Updated editions will replace the previous one—the old editions will
be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States copyright in
these works, so the Foundation (and you!) can copy and distribute it
in the United States without permission and without paying
copyright royalties. Special rules, set forth in the General Terms of
Use part of this license, apply to copying and distributing Project
Gutenberg™ electronic works to protect the PROJECT GUTENBERG™
concept and trademark. Project Gutenberg is a registered trademark,
and may not be used if you charge for an eBook, except by following
the terms of the trademark license, including paying royalties for use
of the Project Gutenberg trademark. If you do not charge anything
for copies of this eBook, complying with the trademark license is
very easy. You may use this eBook for nearly any purpose such as
creation of derivative works, reports, performances and research.
Project Gutenberg eBooks may be modified and printed and given
away—you may do practically ANYTHING in the United States with
eBooks not protected by U.S. copyright law. Redistribution is subject
to the trademark license, especially commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE
 PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK

To protect the Project Gutenberg™ mission of promoting the free

distribution of electronic works, by using or distributing this work (or
any other work associated in any way with the phrase “Project
Gutenberg”), you agree to comply with all the terms of the Full
Project Gutenberg™ License available with this file or online at
www.gutenberg.org/license.

Section 1. General Terms of Use and

Redistributing Project Gutenberg™
electronic works
1.A. By reading or using any part of this Project Gutenberg™
electronic work, you indicate that you have read, understand, agree
to and accept all the terms of this license and intellectual property
(trademark/copyright) agreement. If you do not agree to abide by all
the terms of this agreement, you must cease using and return or
destroy all copies of Project Gutenberg™ electronic works in your
possession. If you paid a fee for obtaining a copy of or access to a
Project Gutenberg™ electronic work and you do not agree to be
bound by the terms of this agreement, you may obtain a refund
from the person or entity to whom you paid the fee as set forth in
paragraph 1.E.8.

1.B. “Project Gutenberg” is a registered trademark. It may only be

used on or associated in any way with an electronic work by people
who agree to be bound by the terms of this agreement. There are a
few things that you can do with most Project Gutenberg™ electronic
works even without complying with the full terms of this agreement.
See paragraph 1.C below. There are a lot of things you can do with
Project Gutenberg™ electronic works if you follow the terms of this
agreement and help preserve free future access to Project
Gutenberg™ electronic works. See paragraph 1.E below.
1.C. The Project Gutenberg Literary Archive Foundation (“the
Foundation” or PGLAF), owns a compilation copyright in the
collection of Project Gutenberg™ electronic works. Nearly all the
individual works in the collection are in the public domain in the
United States. If an individual work is unprotected by copyright law
in the United States and you are located in the United States, we do
not claim a right to prevent you from copying, distributing,
performing, displaying or creating derivative works based on the
work as long as all references to Project Gutenberg are removed. Of
course, we hope that you will support the Project Gutenberg™
mission of promoting free access to electronic works by freely
sharing Project Gutenberg™ works in compliance with the terms of
this agreement for keeping the Project Gutenberg™ name associated
with the work. You can easily comply with the terms of this
agreement by keeping this work in the same format with its attached
full Project Gutenberg™ License when you share it without charge
with others.

1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside the
United States, check the laws of your country in addition to the
terms of this agreement before downloading, copying, displaying,
performing, distributing or creating derivative works based on this
work or any other Project Gutenberg™ work. The Foundation makes
no representations concerning the copyright status of any work in
any country other than the United States.

1.E. Unless you have removed all references to Project Gutenberg:

1.E.1. The following sentence, with active links to, or other

immediate access to, the full Project Gutenberg™ License must
appear prominently whenever any copy of a Project Gutenberg™
work (any work on which the phrase “Project Gutenberg” appears,
or with which the phrase “Project Gutenberg” is associated) is
accessed, displayed, performed, viewed, copied or distributed:
 This eBook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
or re-use it under the terms of the Project Gutenberg License
included with this eBook or online at www.gutenberg.org. If you
are not located in the United States, you will have to check the
laws of the country where you are located before using this
eBook.

1.E.2. If an individual Project Gutenberg™ electronic work is derived

from texts not protected by U.S. copyright law (does not contain a
notice indicating that it is posted with permission of the copyright
holder), the work can be copied and distributed to anyone in the
United States without paying any fees or charges. If you are
redistributing or providing access to a work with the phrase “Project
Gutenberg” associated with or appearing on the work, you must
comply either with the requirements of paragraphs 1.E.1 through
1.E.7 or obtain permission for the use of the work and the Project
Gutenberg™ trademark as set forth in paragraphs 1.E.8 or 1.E.9.

1.E.3. If an individual Project Gutenberg™ electronic work is posted

with the permission of the copyright holder, your use and distribution
must comply with both paragraphs 1.E.1 through 1.E.7 and any
additional terms imposed by the copyright holder. Additional terms
will be linked to the Project Gutenberg™ License for all works posted
with the permission of the copyright holder found at the beginning
of this work.

1.E.4. Do not unlink or detach or remove the full Project

Gutenberg™ License terms from this work, or any files containing a
part of this work or any other work associated with Project
Gutenberg™.

1.E.5. Do not copy, display, perform, distribute or redistribute this

electronic work, or any part of this electronic work, without
prominently displaying the sentence set forth in paragraph 1.E.1
with active links or immediate access to the full terms of the Project
Gutenberg™ License.

1.E.6. You may convert to and distribute this work in any binary,
compressed, marked up, nonproprietary or proprietary form,
including any word processing or hypertext form. However, if you
provide access to or distribute copies of a Project Gutenberg™ work
in a format other than “Plain Vanilla ASCII” or other format used in
the official version posted on the official Project Gutenberg™ website
(www.gutenberg.org), you must, at no additional cost, fee or
expense to the user, provide a copy, a means of exporting a copy, or
a means of obtaining a copy upon request, of the work in its original
“Plain Vanilla ASCII” or other form. Any alternate format must
include the full Project Gutenberg™ License as specified in
paragraph 1.E.1.

1.E.7. Do not charge a fee for access to, viewing, displaying,

performing, copying or distributing any Project Gutenberg™ works
unless you comply with paragraph 1.E.8 or 1.E.9.

1.E.8. You may charge a reasonable fee for copies of or providing

access to or distributing Project Gutenberg™ electronic works
provided that:

• You pay a royalty fee of 20% of the gross profits you derive
from the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
 about donations to the Project Gutenberg Literary Archive
Foundation.”

• You provide a full refund of any money paid by a user who

notifies you in writing (or by e-mail) within 30 days of receipt
that s/he does not agree to the terms of the full Project
Gutenberg™ License. You must require such a user to return or
destroy all copies of the works possessed in a physical medium
and discontinue all use of and all access to other copies of
Project Gutenberg™ works.

• You provide, in accordance with paragraph 1.F.3, a full refund of

any money paid for a work or a replacement copy, if a defect in
the electronic work is discovered and reported to you within 90
days of receipt of the work.

• You comply with all other terms of this agreement for free
distribution of Project Gutenberg™ works.

1.E.9. If you wish to charge a fee or distribute a Project Gutenberg™

electronic work or group of works on different terms than are set
forth in this agreement, you must obtain permission in writing from
the Project Gutenberg Literary Archive Foundation, the manager of
the Project Gutenberg™ trademark. Contact the Foundation as set
forth in Section 3 below.

1.F.

1.F.1. Project Gutenberg volunteers and employees expend

considerable effort to identify, do copyright research on, transcribe
and proofread works not protected by U.S. copyright law in creating
the Project Gutenberg™ collection. Despite these efforts, Project
Gutenberg™ electronic works, and the medium on which they may
be stored, may contain “Defects,” such as, but not limited to,
incomplete, inaccurate or corrupt data, transcription errors, a
copyright or other intellectual property infringement, a defective or
damaged disk or other medium, a computer virus, or computer
codes that damage or cannot be read by your equipment.

1.F.2. LIMITED WARRANTY, DISCLAIMER OF DAMAGES - Except for

the “Right of Replacement or Refund” described in paragraph 1.F.3,
the Project Gutenberg Literary Archive Foundation, the owner of the
Project Gutenberg™ trademark, and any other party distributing a
Project Gutenberg™ electronic work under this agreement, disclaim
all liability to you for damages, costs and expenses, including legal
fees. YOU AGREE THAT YOU HAVE NO REMEDIES FOR
NEGLIGENCE, STRICT LIABILITY, BREACH OF WARRANTY OR
BREACH OF CONTRACT EXCEPT THOSE PROVIDED IN PARAGRAPH
1.F.3. YOU AGREE THAT THE FOUNDATION, THE TRADEMARK
OWNER, AND ANY DISTRIBUTOR UNDER THIS AGREEMENT WILL
NOT BE LIABLE TO YOU FOR ACTUAL, DIRECT, INDIRECT,
CONSEQUENTIAL, PUNITIVE OR INCIDENTAL DAMAGES EVEN IF
YOU GIVE NOTICE OF THE POSSIBILITY OF SUCH DAMAGE.

1.F.3. LIMITED RIGHT OF REPLACEMENT OR REFUND - If you

discover a defect in this electronic work within 90 days of receiving
it, you can receive a refund of the money (if any) you paid for it by
sending a written explanation to the person you received the work
from. If you received the work on a physical medium, you must
return the medium with your written explanation. The person or
entity that provided you with the defective work may elect to provide
a replacement copy in lieu of a refund. If you received the work
electronically, the person or entity providing it to you may choose to
give you a second opportunity to receive the work electronically in
lieu of a refund. If the second copy is also defective, you may
demand a refund in writing without further opportunities to fix the
problem.

1.F.4. Except for the limited right of replacement or refund set forth
in paragraph 1.F.3, this work is provided to you ‘AS-IS’, WITH NO
OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED,
INCLUDING BUT NOT LIMITED TO WARRANTIES OF
MERCHANTABILITY OR FITNESS FOR ANY PURPOSE.

1.F.5. Some states do not allow disclaimers of certain implied

warranties or the exclusion or limitation of certain types of damages.
If any disclaimer or limitation set forth in this agreement violates the
law of the state applicable to this agreement, the agreement shall be
interpreted to make the maximum disclaimer or limitation permitted
by the applicable state law. The invalidity or unenforceability of any
provision of this agreement shall not void the remaining provisions.

1.F.6. INDEMNITY - You agree to indemnify and hold the Foundation,

the trademark owner, any agent or employee of the Foundation,
anyone providing copies of Project Gutenberg™ electronic works in
accordance with this agreement, and any volunteers associated with
the production, promotion and distribution of Project Gutenberg™
electronic works, harmless from all liability, costs and expenses,
including legal fees, that arise directly or indirectly from any of the
following which you do or cause to occur: (a) distribution of this or
any Project Gutenberg™ work, (b) alteration, modification, or
additions or deletions to any Project Gutenberg™ work, and (c) any
Defect you cause.

Section 2. Information about the Mission

of Project Gutenberg™
Project Gutenberg™ is synonymous with the free distribution of
electronic works in formats readable by the widest variety of
computers including obsolete, old, middle-aged and new computers.
It exists because of the efforts of hundreds of volunteers and
donations from people in all walks of life.

Volunteers and financial support to provide volunteers with the

assistance they need are critical to reaching Project Gutenberg™’s
goals and ensuring that the Project Gutenberg™ collection will
remain freely available for generations to come. In 2001, the Project
Gutenberg Literary Archive Foundation was created to provide a
secure and permanent future for Project Gutenberg™ and future
generations. To learn more about the Project Gutenberg Literary
Archive Foundation and how your efforts and donations can help,
see Sections 3 and 4 and the Foundation information page at
www.gutenberg.org.

Section 3. Information about the Project

Gutenberg Literary Archive Foundation
The Project Gutenberg Literary Archive Foundation is a non-profit
501(c)(3) educational corporation organized under the laws of the
state of Mississippi and granted tax exempt status by the Internal
Revenue Service. The Foundation’s EIN or federal tax identification
number is 64-6221541. Contributions to the Project Gutenberg
Literary Archive Foundation are tax deductible to the full extent
permitted by U.S. federal laws and your state’s laws.

The Foundation’s business office is located at 809 North 1500 West,

Salt Lake City, UT 84116, (801) 596-1887. Email contact links and up
to date contact information can be found at the Foundation’s website
and official page at www.gutenberg.org/contact

Section 4. Information about Donations to

the Project Gutenberg Literary Archive
Foundation
Project Gutenberg™ depends upon and cannot survive without
widespread public support and donations to carry out its mission of
increasing the number of public domain and licensed works that can
be freely distributed in machine-readable form accessible by the
widest array of equipment including outdated equipment. Many
small donations ($1 to $5,000) are particularly important to
maintaining tax exempt status with the IRS.

The Foundation is committed to complying with the laws regulating

charities and charitable donations in all 50 states of the United
States. Compliance requirements are not uniform and it takes a
considerable effort, much paperwork and many fees to meet and
keep up with these requirements. We do not solicit donations in
locations where we have not received written confirmation of
compliance. To SEND DONATIONS or determine the status of
compliance for any particular state visit www.gutenberg.org/donate.

While we cannot and do not solicit contributions from states where

we have not met the solicitation requirements, we know of no
prohibition against accepting unsolicited donations from donors in
such states who approach us with offers to donate.

International donations are gratefully accepted, but we cannot make

any statements concerning tax treatment of donations received from
outside the United States. U.S. laws alone swamp our small staff.

Please check the Project Gutenberg web pages for current donation
methods and addresses. Donations are accepted in a number of
other ways including checks, online payments and credit card
donations. To donate, please visit: www.gutenberg.org/donate.

Section 5. General Information About

Project Gutenberg™ electronic works
Professor Michael S. Hart was the originator of the Project
Gutenberg™ concept of a library of electronic works that could be
freely shared with anyone. For forty years, he produced and
distributed Project Gutenberg™ eBooks with only a loose network of
volunteer support.
Project Gutenberg™ eBooks are often created from several printed
editions, all of which are confirmed as not protected by copyright in
the U.S. unless a copyright notice is included. Thus, we do not
necessarily keep eBooks in compliance with any particular paper
edition.

Most people start at our website which has the main PG search
facility: www.gutenberg.org.

This website includes information about Project Gutenberg™,

including how to make donations to the Project Gutenberg Literary
Archive Foundation, how to help produce our new eBooks, and how
to subscribe to our email newsletter to hear about new eBooks.
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

textbookfull.com