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Classification of Microbiota –

An Overview of Clinical Microbiology, Classification, and Antimicrobial Resistance

Author and Curator: Larry H. Bernstein, MD, FCAP

Classification of Microbiota

Introduction to Overview of Microbiology

This is a contribution to a series of pieces on the history of biochemistry, molecular biology, physiology and medicine in the 20th century.  Here I describe the common microbial organisms encountered in the clinical laboratory, the method of their collection, plating, culture and identification, and antibiotic sensitivity testing and resistant strains.

I may begin with the recognition that there are common strains in the environment that are not pathogenic, and there are pathogenic bacteria.
In addition, there are bacteria that coexist in the body habitat under specific conditions so that we are able to map the types expected to location, such as, skin, mouth and nasal cavities, the colon, the vagina and urinary system.  Meningitides occur as a result of extension from the nasal cavity to the brain.  When bacteria invade the circulation, it is referred to as septicemia, and the bacteria can cause valvular heart damage.

Bacteriology can be traced to origins in the 19th century.  The clinical features of localized infection are classically referred to as redness, heat, a raised lesion (pustule), and exudate (serous or purulent – watery or cellular).  This not only holds for a focal lesion (as skin), but also for pneumonia, urinary infection, and genital. It may be accompanied by cough, or bloody cough and wheezing, or by an unclear urine. In the case of septicemia, there is fever, and there may be seizures or delirium.

Collection and handling of specimens

Specimens are collected by sterile technique by a nurse or physician and sent to a lab as a swab, or as a blood specimen.  In the case of a febrile illness, blood cultures may be obtained from opposite arms, and another an hour later.  This is related to the possible cyclical seeding of bacteria into the circulation.  If the specimen is collected from a site of infection, a swab may be put onto a glass slide for gram staining.  The specimen collected is sent to the laboratory.

We may consider syphilis and tuberculosis special cases that I’ll set aside.  I shall not go into virology either, although I may referred to smallpox, influenza, polio, HIV under epidemic.  The first step in identification is the Gram stain, developed in the 19th century.  Organisms of the skin are Gram positive and appear blue on staining.  They are cocci, or circular, organized in characteristic clusters (staphylococcus, streptococcus) or in pairs (diplococci, eg. Pneumococcus), and if from the intestine (enterococcus).  If they are elongated rods, they might be coliform.  If they stain red, they are Gram negative.  Gram negative rods are coliform, and are enterobacteriaceae. Meningococci are Gram negative cocci.  So we have certain information about these organisms before we plate them for growth.

Laboratory growth characteristics

The specimen is applied to an agar plate with a metal rod applicator, or perhaps onto more than one agar plate.  The agar plate contains a growth media or a growth inhibitor that is more favorable to certain species than to others.  The bacteria are grown at 37o C in an incubator and colonies develop that are white or nonwhite, and they are smooth or wrinkled.  The appearance of the colonies is characteristic for certain strains.  If there is no contamination, all of the colonies look the same.  The next step is to:

  • Gram stain from a colony
  • Transfer samples from the colony to a series of growth media that identify presence or absence of specific nutrient requirements for growth (which is presumed from the prior findings).

In addition, the colony samples are grown on an agar to which is applied antibiotic tabs.  The tabs either allow or repress growth.  It wa some 50 years ago that the infectious disease physician and microbiologist Abraham Braude would culture the bacteria on agar plates that had a gradient of antibiotic to check for concentration that would inhibit growth.

Principles of Diagnosis (Extracts)

By John A. Washington

The clinical presentation of an infectious disease reflects the interaction between the host and the microorganism. This interaction is affected by the host immune status and microbial virulence factors. Signs and symptoms vary according to the site and severity of infection. Diagnosis requires a composite of information, including history, physical examination, radiographic findings, and laboratory data.

Microbiologic Examination

Direct Examination and Techniques: Direct examination of specimens reveals gross pathology. Microscopy may identify microorganisms. Immunofluorescence, immuno-peroxidase staining, and other immunoassays may detect specific microbial antigens. Genetic probes identify genus- or species-specific DNA or RNA sequences.

Culture: Isolation of infectious agents frequently requires specialized media. Nonselective (noninhibitory) media permit the growth of many microorganisms. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms.

Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic probes are also used.

Serodiagnosis: A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis.

Antimicrobial Susceptibility: Microorganisms, particularly bacteria, are tested in vitro to determine whether they are susceptible to antimicrobial agents.

Diagnostic medical microbiology is the discipline that identifies etiologic agents of disease. The job of the clinical microbiology laboratory is to test specimens from patients for microorganisms that are, or may be, a cause of the illness and to provide information (when appropriate) about the in vitro activity of antimicrobial drugs against the microorganisms identified (Fig. 1).

Laboratory procedures used in confirming a clinical diagnosis of infectious disease with a bacterial etiology

A variety of microscopic, immunologic, and hybridization techniques have been developed for rapid diagnosis

techniques have been developed for rapid diagnosis

techniques have been developed for rapid diagnosis

From: Chapter 10, Principles of Diagnosis
Medical Microbiology. 4th edition.
Baron S, editor.
Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

For immunologic detection of microbial antigens, latex particle agglutination, coagglutination, and enzyme-linked immunosorbent assay (ELISA) are the most frequently used techniques in the clinical laboratory. Antibody to a specific antigen is bound to latex particles or to a heat-killed and treated protein A-rich strain of Staphylococcus aureus to produce agglutination (Fig. 10-2). There are several approaches to ELISA; the one most frequently used for the detection of microbial antigens uses an antigen-specific antibody that is fixed to a solid phase, which may be a latex or metal bead or the inside surface of a well in a plastic tray. Antigen present in the specimen binds to the antibody as inFig. 10-2. The test is then completed by adding a second antigen-specific antibody bound to an enzyme that can react with a substrate to produce a colored product. The initial antigen antibody complex forms in a manner similar to that shown inFigure 10-2. When the enzyme-conjugated antibody is added, it binds to previously unbound antigenic sites, and the antigen is, in effect, sandwiched between the solid phase and the enzyme-conjugated antibody. The reaction is completed by adding the enzyme substrate.

agglutination test ch10f2

agglutination test ch10f2

Figure 2 Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria

Genetic probes are based on the detection of unique nucleotide sequences with the DNA or RNA of a microorganism. Once such a unique nucleotide sequence, which may represent a portion of a virulence gene or of chromosomal DNA, is found, it is isolated and inserted into a cloning vector (plasmid), which is then transformed into Escherichia coli to produce multiple copies of the probe. The sequence is then reisolated from plasmids and labeled with an isotope or substrate for diagnostic use. Hybridization of the sequence with a complementary sequence of DNA or RNA follows cleavage of the double-stranded DNA of the microorganism in the specimen.

The use of molecular technology in the diagnoses of infectious diseases has been further enhanced by the introduction of gene amplication techniques, such as the polymerase chain reaction (PCR) in which DNA polymerase is able to copy a strand of DNA by elongating complementary strands of DNA that have been initiated from a pair of closely spaced oligonucleotide primers. This approach has had major applications in the detection of infections due to microorganisms that are difficult to culture (e.g. the human immunodeficiency virus) or that have not as yet been successfully cultured (e.g. the Whipple’s disease bacillus).

Solid media, although somewhat less sensitive than liquid media, provide isolated colonies that can be quantified if necessary and identified. Some genera and species can be recognized on the basis of their colony morphologies.

In some instances one can take advantage of differential carbohydrate fermentation capabilities of microorganisms by incorporating one or more carbohydrates in the medium along with a suitable pH indicator. Such media are called differential media (e.g., eosin methylene blue or MacConkey agar) and are commonly used to isolate enteric bacilli. Different genera of the Enterobacteriaceae can then be presumptively identified by the color as well as the morphology of colonies.

Culture media can also be made selective by incorporating compounds such as antimicrobial agents that inhibit the indigenous flora while permitting growth of specific microorganisms resistant to these inhibitors. One such example is Thayer-Martin medium, which is used to isolate Neisseria gonorrhoeae. This medium contains vancomycin to inhibit Gram-positive bacteria, colistin to inhibit most Gram-negative bacilli, trimethoprim-sulfamethoxazole to inhibit Proteus species and other species that are not inhibited by colistin and anisomycin to inhibit fungi. The pathogenic Neisseria species, N gonorrhoeae and N meningitidis, are ordinarily resistant to the concentrations of these antimicrobial agents in the medium.

Infection of the bladder (cystitis) or kidney (pyelone-phritis) is usually accompanied by bacteriuria of about ≥ 104 CFU/ml. For this reason, quantitative cultures (Fig. 10-3) of urine must always be performed. For most other specimens a semiquantitative streak method (Fig. 10-3) over the agar surface is sufficient. For quantitative cultures, a specific volume of specimen is spread over the agar surface and the number of colonies per milliliter is estimated.

Identification of bacteria (including mycobacteria) is based on growth characteristics (such as the time required for growth to appear or the atmosphere in which growth occurs), colony and microscopic morphology, and biochemical, physiologic, and, in some instances, antigenic or nucleotide sequence characteristics. The selection and number of tests for bacterial identification depend upon the category of bacteria present (aerobic versus anaerobic, Gram-positive versus Gram-negative, cocci versus bacilli) and the expertise of the microbiologist examining the culture. Gram-positive cocci that grow in air with or without added CO2 may be identified by a relatively small number of tests. The identification of most Gram-negative bacilli is far more complex and often requires panels of 20 tests for determining biochemical and physiologic characteristics.

Antimicrobial susceptibility tests are performed by either disk diffusion or a dilution method. In the former, a standardized suspension of a particular microorganism is inoculated onto an agar surface to which paper disks containing various antimicrobial agents are applied. Following overnight incubation, any zone diameters of inhibition about the disks are measured. An alternative method is to dilute on a log2 scale each antimicrobial agent in broth to provide a range of concentrations and to inoculate each tube or, if a microplate is used, each well containing the antimicrobial agent in broth with a standardized suspension of the microorganism to be tested. The lowest concentration of antimicrobial agent that inhibits the growth of the microorganism is the minimal inhibitory concentration.

Classification Principles

This Week’s Citation Classic®_______ Sneath P H A & Sokal R R.
Numerical taxonomy: the principles and practice of
numerical classification. San Francisco: Freeman, 1973. 573 p.
[Medical Research Council Microbial Systematics Unit, Univ. Leicester, England
and Dept. Ecology and Evolution, State Univ. New York, Stony Brook, NY]
Numerical taxonomy establishes classification
of organisms based on their similarities. It utilizes
many equally weighted characters and employs
clustering and similar algorithms to yield
objective groupings. It can beextended to give
phylogenetic or diagnostic systems and can be
applied to many other fields of endeavour.

Mathematical Foundations of Computer Science 1998
Lecture Notes in Computer Science Volume 1450, 1998, pp 474-482
Date: 28 May 2006
Positive Turing and truth-table completeness for NEXP are incomparable 1998
Levke Bentzien

The truth-table method [matrix method] is one of the decision procedures for sentence logic (q.v., §3.2). The method is based on the fact that the truth value of a compound formula of sentence logic, construed as a truth-function, is determined by the truth values of its arguments (cf. “Sentence logic” §2.2). To decide whether a formula A is a tautology or not, we list all possible combinations of truth values to the variables in A: A is a tautology if it takes the value truth under each assignment.

Using ideas introduced by Buhrman et al. ([2], [3]) to separate various completeness notions for NEXP = NTIME (2poly), positive Turing complete sets for NEXP are studied. In contrast to many-one completeness and bounded truth-table completeness with norm 1 which are known to coincide on NEXP ([3]), whence any such set for NEXP is positive Turing complete, we give sets A and B such that

A is ≤ bT(2) P -complete but not ≤ posT P -complete for NEXP

B is ≤ posT P -complete but not ≤ tt P -complete for NEXP. These results come close to optimality since a further strengthening of (1), as was done by Buhrman in [1] for EXP = DTIME(2poly), seems to require the assumption NEXP = co-NEXP.

Computability and Models
The University Series in Mathematics 2003, pp 1-10
Truth-Table Complete Computably Enumerable Sets
Marat M. Arslanov

We prove a truth-table completeness criterion for computably enumerable sets.
The authors research was partially supported by Russian Foundation of Basic Research, Project 99-01-00830, and RFBR-INTAS, Project 97-91-71991.

Department of Microbiology, Lovelace Foundation for Medical Education and Research,
Albuquerque, N.M. 87108, U.S.A.
Space life sciences 1971-12-1; 3(2): pp 135-156
(Received 15 July, 1971)
Abstract. A logical basis for classification is that elements grouped together and higher categories of elements should have a high degree of similarity with the provision that all groups and categories be disjoint to some degree. A methodology has been developed for constructing classifications automatically that gives
nearly instantaneous correlations of character patterns of organisms with time and clusters with apparent similarity. This means that automatic numerical identification will always construct schemes from which disjoint answers can be obtained if test sensitivities for characters are correct. Unidentified organisms are recycled through continuous classification with reconstruction of identification schemes. This process is
cyclic and self-correcting. The method also accumulates and analyzes data which updates and presents a more accurate biological picture.

Syndromic classification: A process for amplifying information using S-clustering

Eugene W. Rypka, PHD

Optimal classification/Rypka < Optimal classification>


1 Rypka’s Method

1.1 Equations

1.2 Examples

2 Notes and References

Rypka’s Method

Rypka’s[1] method[2] utilizes the theoretical and empirical separatory equations shown below to perform the task of optimal classification. The method finds the optimal order of the fewest attributes, which in combination define a bounded class of elements.

Application of the method begins with construction of an attribute-valued system in truth table[3] or spreadsheet form with elements listed in the left most column beginning in the second row. Characteristics[4] are listed in the first row beginning in the second column with the code name of the data in the upper left most cell. The values which connect each characteristic with each element are placed in the intersecting cells. Selecting appropriate characteristics to universally define the class of elements may be the most difficult part for the classifier of utilizing this method.

The elements are first sorted in descending order according to their truth table value, which is calculated from the existing sequence and value of characteristics for each element. Duplicate truth table values or multisets for the entire bounded class reveal either the need to eliminate duplicate elements or the need to include additional characteristics.

An empirical separatory value is calculated for each characteristic in the set and the characteristic with the greatest empirical separatory value is exchanged with the characteristic which occupies the most significant attribute position.

Next the second most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first characteristic. The characteristic which produces the greatest separatory value is then exchanged with the characteristic which occupies the second most significant attribute position.

Next the third most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first and second characteristics. The characteristic which produces the greatest empirical separatory value is then exchanged with the characteristic which occupies the third most significant attribute position. This procedure may continue until all characteristics have been processed or until one hundred percent separation of the elements has been achieved.

A larger radix will allow faster identification by excluding a greater percentage of elements per characteristic. A binary radix for instance excludes only fifty percent of the elements per characteristic whereas a five-valued radix excludes eighty percent of the elements per characteristic.[5] What follows is an elucidation of the matrix and separatory equations.[6]

Computational Example
Bounded Class Data

bounded class data

Bounded Class Dimensions

G = 28 – 28 elements – i = 0…G-1[1]

C = 10 – 10 characteristics or attributes – j = 0…C-1

V = 5 – 5 valued logic – l = 0…V-1

Order of Elements

order of elements

Count multisets

count multisets

Squared multiset Counts

squared multiset counts

Separatory Values

separatory values


max(T) = 309 = S8 = highest initial separatory value


Mathcad’s ORIGIN function applies to all arrays such that if more than one array is being used and one array requires a zero origin then the other arrays must use a zero origin with all variables being adapted as well.

Rypka’s Method Edit

Rypka’s[1] method[2] utilizes the theoretical and empirical separatory equations shown below to perform the task of optimal classification. The method finds the optimal order of the fewest attributes, which in combination define a bounded class of elements.

Application of the method begins with construction of an attribute-valued system in truth table[3] or spreadsheet form with elements listed in the left most column beginning in the second row. Characteristics[4] are listed in the first row beginning in the second column with the title of the attributes in the upper left most cell. Normally the file name of the data is given the title of the element class. The values which connect each characteristic with each element are placed in the intersecting cells. Selecting characteristics which all elements share may be the most difficult part of creating a database which can utilizing this method.

The elements are first sorted in descending order according to their truth table value, which is calculated from the existing sequence and value of characteristics for each element. Duplicate truth table values or multisets for the entire bounded class reveal either the need to eliminate duplicate elements or the need to include additional characteristics.

An empirical separatory value is calculated for each characteristic in the set and the characteristic with the greatest empirical separatory value is exchanged with the characteristic which occupies the most significant attribute position.

Next the second most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first characteristic. The characteristic which produces the greatest separatory value is then exchanged with the characteristic which occupies the second most significant attribute position.

Next the third most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first and second characteristics. The characteristic which produces the greatest empirical separatory value is then exchanged with the characteristic which occupies the third most significant attribute position. This procedure may continue until all characteristics have been processed or until one hundred percent separation of the elements has been achieved.

A larger radix will allow faster identification by excluding a greater percentage of elements per characteristic. A binary radix for instance excludes only fifty percent of the elements per characteristic whereas a five-valued radix excludes eighty percent of the elements per characteristic.[5] What follows is an elucidation of the matrix and separatory equations.[6]

Syndromic Classification: A Process for Amplifying Information Using S-Clustering

Eugene W. Rypka, PhD
University of New Mexico, Albuquerque, New Mexico, USA
Statistics Editor: Marcello Pagano, PhD
Harvard School of Public Health, Boston, Massachusetts, USA
Nutrition 1996; 12(11/12): 827-829

In a previous issue of Nutrition, Drs. Bernstein and Pleban’ use the method of S-clustering to aid in nutritional classification of patients directly on-line. Classification of this type is called primary or syndromic classification.* It is created by a process called separatory (S-) clustering (E. Rypka, unpublished observations). The authors use S-clustering in Table I.  S-clustering extracts features (analytes, variables) from endogenous data that amplify or maximize structural information to create classes of patients (pathophysiologic events) which are the most disjointed or separable. S-clustering differs from other classificatory methods because it finds in a database a theoretic- or more- number of variables with the required variety that map closest to an ideal, theoretic, or structural information standard. In Table I of their article, Bernstein and Pleban’ indicate there would have to be 3 ’ = 243 rows to show all possible patterns. In Table II of this article, I have used a 33 = 27 row truth table to convey the notion of mapping amplified information to an ideal, theoretic standard using just the first three columns. Variables are scaled for use in S-clustering.

A Survey of Binary Similarity and Distance Measures
Seung-Seok Choi, Sung-Hyuk Cha, Charles C. Tappert
The binary feature vector is one of the most common
representations of patterns and measuring similarity and
distance measures play a critical role in many problems
such as clustering, classification, etc. Ever since Jaccard
proposed a similarity measure to classify ecological
species in 1901, numerous binary similarity and distance
measures have been proposed in various fields. Applying
appropriate measures results in more accurate data
analysis. Notwithstanding, few comprehensive surveys
on binary measures have been conducted. Hence we
collected 76 binary similarity and distance measures used
over the last century and reveal their correlations through
the hierarchical clustering technique.

This paper is organized as follows. Section 2 describes
the definitions of 76 binary similarity and dissimilarity
measures. Section 3 discusses the grouping of those
measures using hierarchical clustering. Section 4
concludes this work.

Historically, all the binary measures observed above have
had a meaningful performance in their respective fields.
The binary similarity coefficients proposed by Peirce,
Yule, and Pearson in 1900s contributes to the evolution
of the various correlation based binary similarity
measures. The Jaccard coefficient proposed at 1901 is
still widely used in the various fields such as ecology and
biology. The discussion of inclusion or exclusion of
negative matches was actively arisen by Sokal & Sneath
in during 1960s and by Goodman & Kruskal in 1970s.

Polyphasic Taxonomy of the Genus Vibrio: Numerical Taxonomy of Vibrio cholerae, Vibrio
parahaemolyticus, and Related Vibrio Species
JOURNAL OF BACTERIOLOGY, Oct. 1970;  104(1): 410-433
A set of 86 bacterial cultures, including 30 strains of Vibrio cholerae, 35 strains of
V. parahaemolyticus, and 21 representative strains of Pseudomonas, Spirillum,
Achromobacter, Arthrobacter, and marine Vibrio species were tested for a total of 200
characteristics. Morphological, physiological, and biochemical characteristics were
included in the analysis. Overall deoxyribonucleic acid (DNA) base compositions
and ultrastructure, under the electron microscope, were also examined. The taxonomic
data were analyzed by computer by using numerical taxonomy programs
designed to sort and cluster strains related phenetically. The V. cholerae strains
formed an homogeneous cluster, sharing overall S values of >75%. Two strains,
V. cholerae NCTC 30 and NCTC 8042, did not fall into the V. cholerae species
group when tested by the hypothetical median organism calculation. No separation
of “classic” V. cholerae, El Tor vibrios, and nonagglutinable vibrios was observed.
These all fell into a single, relatively homogeneous, V. cholerae species cluster.
PJ. parahaemolyticus strains, excepting 5144, 5146, and 5162, designated members
of the species V. alginolyticus, clustered at S >80%. Characteristics uniformly
present in all the Vibrio species examined are given, as are also characteristics and
frequency of occurrence for V. cholerae and V. parahaemolyticus. The clusters formed
in the numerical taxonomy analyses revealed similar overall DNA base compositions,
with the range for the Vibrio species of 40 to 48% guanine plus cytosine. Generic
level of relationship of V. cholerae and V. parahaemolyticus is considered
dubious. Intra- and intergroup relationships obtained from the numerical taxonomy
studies showed highly significant correlation with DNA/DNA reassociation data.

A Numerical Classification of the Genus Bacillus
Journal of General Microbiology (1988), 134, 1847-1882.

Three hundred and sixty-eight strains of aerobic, endospore-forming bacteria which included type and reference cultures of Bacillus and environmental isolates were studied. Overall similarities of these strains for 118 unit characters were determined by the SSMS,, and Dp coefficients and clustering achieved using the UPGMA algorithm. Test error was within acceptable limits. Six cluster-groups were defined at 70% SSM which corresponded to 69% Sp and 48-57% SJ.G roupings obtained with the three coefficients were generally similar but there were some changes in the definition and membership of cluster-groups and clusters, particularly with the SJ coefficient. The Bacillus strains were distributed among 31 major (4 or more strains), 18 minor (2 or 3 strains) and 30 single-member clusters at the 83% SsMle vel. Most of these clusters can be regarded as taxospecies. The heterogeneity of several species, including Bacillus breuis, B. circulans, B. coagulans, B. megateriun, B . sphaericus and B . stearothermophilus, has been indicated  and the species status of several taxa of hitherto uncertain validity confirmed. Thus on the basis of the numerical phenetic and appropriate (published) molecular genetic data, it is proposed
that the following names be recognized; BacillusJlexus (Batchelor) nom. rev., Bacillus fusiformis (Smith et al.) comb. nov., Bacillus kaustophilus (Prickett) nom. rev., Bacilluspsychrosaccharolyticus (Larkin & Stokes) nom. rev. and Bacillus simplex (Gottheil) nom. rev. Other phenetically well-defined taxospecies included ‘ B. aneurinolyticus’, ‘B. apiarius’, ‘B. cascainensis’, ‘B. thiaminolyticus’ and three clusters of environmental isolates related to B . firmus and previously described as ‘B. firmus-B. lentus intermediates’. Future developments in the light of the numerical phenetic data are discussed.

Numerical Classification of Bacteria
Part II. * Computer Analysis of Coryneform Bacteria (2)
Comparison of Group-Formations Obtained on Two
Different Methods of Scoring Data
By Eitaro MASUOan d Toshio NAKAGAWA
[Agr. Biol. Chem., 1969; 33(8): 1124-1133.
Sixty three organisms selected from 12 genera of bacteria were subjected to numerical analysis. The purpose of this work is to examine the relationships among 38 coryneform bacteria included in the test organisms by two coding methods-Sneath’s and Lockhart’s systems-, and to compare the results with conventional classification. In both cases of codification, five groups and one or two single item(s) were found in the resultant classifications. Different codings brought, however, a few distinct differences in some groups , especially in a group of sporogenic bacilli or lactic-acid bacteria. So far as the present work concerns, the result obtained on Lockhart’s coding rather than that obtained on Sneath’s coding resembled the conventional classification. The taxonomic positions of corynebacteria were quite different from those of the conventional classification, regardless
of which coding method was applied.
Though animal corynebacteria have conventionally been considered to occupy the
taxonomic position neighboring to genera Arthrobacter and Cellulornonas and regarded to be the nucleus of so-called “coryneform bacteria,’ the present work showed that many of the corynebacteria are akin to certain mycobacteria rather than to the organisms belonging to the above two genera.

Numerical Classification of Bacteria
Part III. Computer Analysis of “Coryneform Bacteria” (3)
Classification Based on DNA Base Compositions
By EitaroM ASUaOnd ToshioN AKAGAWA
Agr. Biol. Chem., 1969; 33(11): 1570-1576
It has been known that the base compositions of deoxyribonucleic acids (DNA) are
quite different from organism to organism. A pertinent example of this diversity is
found in bacterial species. The base compositions of DNA isolated from a wide variety
of bacteria are distributed in a range from 25 to 75 GC mole-percent (100x(G+C)/
(A+T+G+C)).1) The usefulness of the information of DNA base composition for
the taxonomy of bacteria has been emphasized by several authors. Lee et al.,” Sueoka,” and Freese) have speculated on the evolutionary significance of microbial DNA base composition. They pointed out that closely related microorganisms generally showed similar base compositions of DNA, and suggested that phylogenetic relationship should be reflected in the GC content.
In the present paper are compared the results of numerical classifications of 45
bacteria based on the two different similarity matrices: One representing the overall
similarities of phenotypic properties, the other representing the similarities of GC contents.

Advanced computational algorithms for microbial community analysis using massive 16S rRNA
sequence data
Y Sun, Y Cai, V Mai, W Farmerie, F Yu, J Li and S Goodison
Nucleic Acids Research, 2010; 38(22): e205

With the aid of next-generation sequencing technology, researchers can now obtain millions of microbial signature sequences for diverse applications ranging from human epidemiological studies to global ocean surveys. The development of advanced computational strategies to maximally extract pertinent information from massive nucleotide data has become a major focus of the bioinformatics community. Here, we describe a novel analytical strategy including discriminant and topology analyses that enables researchers to deeply investigate the hidden world of microbial communities, far beyond basic microbial diversity estimation. We demonstrate the utility of our
approach through a computational study performed on a previously published massive human gut 16S rRNA data set. The application of discriminant and
topology analyses enabled us to derive quantitative disease-associated microbial signatures and describe microbial community structure in far more detail than previously achievable. Our approach provides rigorous statistical tools for sequence based studies aimed at elucidating associations between known or unknown organisms and a variety of physiological or environmental conditions.

What is Drug Resistance?

Antimicrobial resistance is the ability of microbes, such as bacteria, viruses, parasites, or fungi, to grow in the presence of a chemical (drug) that would normally kill it or limit its growth.

Diagram showing the difference between non-resistant bacteria and drug resistant bacteria.

Credit: NIAID

DrugResistance difference between non-resistant bacteria and drug resistant bacteria

DrugResistance difference between non-resistant bacteria and drug resistant bacteria

Diagram showing the difference between non-resistant bacteria and drug resistant bacteria. Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug resistant bacteria multiply as well, but upon drug treatment, the bacteria continue to spread.

Between 5 and 10 percent of all hospital patients develop an infection. About 90,000 of these patients die each year as a result of their infection, up from 13,300 patient deaths in 1992.

According to the Centers for Disease Control and Prevention (April 2011), antibiotic resistance in the United States costs an estimated $20 billion a year in excess health care costs, $35 million in other societal costs and more than 8 million additional days that people spend in the hospital.

Resistance to Antibiotics: Are We in the Post-Antibiotic Era?

Alfonso J. Alanis
Archives of Medical Research 36 (2005) 697–705

Serious infections caused by bacteria that have become resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. They not only are more severe and require longer and more complex treatments, but they are also significantly more expensive to diagnose and to treat. Antibiotic resistance, initially a problem of the hospital setting associated with an increased number of hospital acquired infections usually in critically ill and immunosuppressed patients, has now extended into the community causing severe infections difficult to diagnose and treat. The molecular mechanisms by which bacteria have become resistant to antibiotics are diverse and complex. Bacteria have developed resistance to all different classes of antibiotics discovered to date. The most frequent type of resistance is acquired and transmitted horizontally via the conjugation of a plasmid. In recent times new mechanisms of resistance have resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (MDR) bacterial strains, some also known as ‘‘superbugs’’. The indiscriminate and inappropriate use of antibiotics in outpatient clinics, hospitalized patients and in the food industry is the single largest factor leading to antibiotic resistance. The pharmaceutical industry, large academic institutions or the government are not investing the necessary resources to produce the next generation of newer safe and effective antimicrobial drugs. In many cases, large pharmaceutical companies have terminated their anti-infective research programs altogether due to economic reasons. The potential negative consequences of all these events are relevant because they put society at risk for the spread of potentially serious MDR bacterial infections.

Targeting the Human Macrophage with Combinations of Drugs and Inhibitors of Ca2+ and K+ Transport to Enhance the Killing of Intracellular Multi-Drug Resistant M. tuberculosis (MDR-TB) – a Novel, Patentable Approach to Limit the Emergence of XDR-TB

Marta Martins
Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

The emergence of resistance in Tuberculosis has become a serious problem for the control of this disease. For that reason, new therapeutic strategies that can be implemented in the clinical setting are urgently needed. The design of new compounds active against mycobacteria must take into account that Tuberculosis is mainly an intracellular infection of the alveolar macrophage and therefore must maintain activity within the host cells. An alternative therapeutic approach will be described in this review, focusing on the activation of the phagocytic cell and the subsequent killing of the internalized bacteria. This approach explores the combined use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors, in the infected macrophage. Targeting the infected macrophage and not the internalized bacteria could overcome the problem of bacterial multi-drug resistance. This will potentially eliminate the appearance of new multi-drug resistant tuberculosis (MDR-TB) cases and subsequently prevent the emergence of extensively-drug resistant tuberculosis (XDR-TB). Patents resulting from this novel and innovative approach could be extremely valuable if they can be implemented in the clinical setting. Other patents will also be discussed such as the treatment of TB using immunomodulator compounds (for example: betaglycans).

Six Epigenetic Faces of Streptococcus

Kevin Mayer

Medical illustration of Streptococcus pneumonia. [CDC]

Streptococcus pneumonia

Streptococcus pneumonia

It appears that S. pneumoniae has even more personalities, each associated with a different proclivity toward invasive, life-threatening disease. In fact, any of six personalities may emerge depending on the action of a single genetic switch.

To uncover the switch, an international team of scientists conducted a study in genomics, but they looked beyond nucleotide polymorphisms or accessory regions as possible phenotype-shifting mechanisms. Instead, they focused on the potential of restriction-modification (RM) systems to mediate gene regulation via epigenetic changes.

Scientists representing the University of Leicester, Griffith University’s Institute for Glycomics, theUniversity of Adelaide, and Pacific Biosciences realized that the S. pneumoniae genome contains two Type I, three Type II, and one Type IV RM systems. Of these, only the DpnI Type II RM system had been described in detail. Switchable Type I systems had been described previously, but these reports did not provide evidence for differential methylation or for phenotypic impact.

As it turned out, the Type I system embodied a mechanism capable of randomly changing the bacterium’s characteristics into six alternative states. The mechanism’s details were presented September 30 in Nature Communications, in an article entitled, “A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.”

“The underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III),” wrote the authors. “The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics.”

Eradication of multidrug-resistant A. baumanniii in burn wounds by antiseptic pulsed electric field.

A Golberg, GF Broelsch, D Vecchio,S Khan, MR Hamblin, WG Austen, Jr, RL Sheridan,  ML Yarmush.

Emerging bacterial resistance to multiple drugs is an increasing problem in burn wound management. New non-pharmacologic interventions are needed for wound disinfection. Here we report on a novel physical method for disinfection: antiseptic pulsed electric field (PEF) applied externally to the infected wounds.  In an animal model, we show that PEF can reduce the load of multidrug resistant Acinetobacter baumannii present in a full thickness burn wound by more than four orders of magnitude, as detected by bioluminescence imaging. Furthermore, using a finite element numerical model, we demonstrate that PEF provides non-thermal, homogeneous, full thickness treatment for the burn wound, thus, overcoming the limitation of treatment depth for many topical antimicrobials. These modeling tools and our in vivo results will be extremely useful for further translation of the PEF technology to the clinical setting. We believe that PEF, in combination with systemic antibiotics, will synergistically eradicate multidrug-resistant burn wound infections, prevent biofilm formation and restore natural skin microbiome. PEF provides a new platform for infection combat in patients, therefore it has a potential to significantly decreasing morbidity and mortality.

Golberg, A. & Yarmush, M. L. Nonthermal irreversible electroporation: fundamentals, applications, and challenges. IEEE Trans Biomed Eng 60, 707-14 (2013).

Mechanisms Of Antibiotic Resistance In Salmonella: Efflux Pumps, Genetics, Quorum Sensing And Biofilm Formation.

Martins M, McCusker M, Amaral L, Fanning S
Perspectives in Drug Discovery and Design 02/2011; 8:114-123.

In Salmonella the main mechanisms of antibiotic resistance are mutations in target genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as changes in the cell envelope; down regulation of membrane porins; increased lipopolysaccharide (LPS) component of the outer cell membrane; quorum sensing and biofilm formation can also contribute to the resistance seen in this microorganism. To overcome this problem new therapeutic approaches are urgently needed. In the case of efflux-mediated multidrug resistant isolates, one of the treatment options could be the use of efflux pump inhibitors (EPIs) in combination with the antibiotics to which the bacteria is resistant. By blocking the efflux pumps resistance is partly or wholly reversed, allowing antibiotics showing no activity against the MDR strains to be used to treat these infections. Compounds that show potential as an EPI are therefore of interest, as well as new strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation are systems also known to be involved in antibiotic resistance. Consequently, compounds that can disrupt or inhibit these bacterial “communication systems” will be of use in the treatment of these infections.

Role of Phenothiazines and Structurally Similar Compounds of Plant Origin in the Fight against Infections by Drug Resistant Bacteria

SG Dastidar, JE Kristiansen, J Molnar and L Amaral
Antibiotics 2013, 2, 58-71;

Phenothiazines have their primary effects on the plasma membranes of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources and energy providing enzymes, such as ATPase, and genes that regulate and code for the permeability aspect of a bacterium. The response of multidrug and extensively drug resistant tuberculosis to phenothiazines shows an alternative therapy for its treatment. Many phenothiazines have shown synergistic activity with several antibiotics thereby lowering the doses of antibiotics administered for specific bacterial infections. Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp) has been found to be synergistic with penicillin and chlorpromazine (CPZ); in addition, some antibiotics are also synergistic. Along with the antibacterial action described in this review, many phenothiazines possess plasmid curing activities, which render the bacterial carrier of the plasmid sensitive to antibiotics. Thus, simultaneous applications of a phenothiazine like TZ would not only act as an additional antibacterial agent but also would help to eliminate drug resistant plasmid from the infectious bacterial cells.

Multidrug Efflux Pumps Described for Staphylococcus aureus

Efflux Pump  Family Regulator(s) Substrate Specificity  References 
Chromosomally-encoded Efflux Systems 
NorA MFS MgrA, NorG(?) Hydrophilic fluoroquinolones (ciprofloxacin, norfloxacin)QACs (tetraphenylphosphonium, benzalkonium chloride)

Dyes (e.g. ethidium bromide, rhodamine)

NorB MFS MgrA, NorG Fluoroquinolones (e.g. hydrophilic: ciprofloxacin, norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin)TetracyclineQACs (e.g. tetraphenylphosphonium, cetrimide)Dyes (e.g. ethidium bromide)
NorC MFS MgrA(?), NorG Fluoroquinolones (e.g. hydrophilic: ciprofloxacin and hydrophobic: moxifloxacin)Dyes (e.g. rhodamine) [35,36]
MepA MATE MepR Fluoroquinolones (e.g. hydrophilic: ciprofloxacin, norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin)Glycylcyclines (e.g. tigecycline)QACs (e.g. tetraphenylphosphonium, cetrimide, benzalkonium chloride)Dyes (e.g. ethidium bromide)
MdeA MFS n.i. Hydrophilic fluoroquinolones (e.g. ciprofloxacin, norfloxacin)Virginiamycin, novobiocin, mupirocin, fusidic acid

QACs (e.g. tetraphenylphosphonium, benzalkonium chloride, dequalinium)

Dyes (e.g. ethidium bromide)

SepA n.d. n.i. QACs (e.g. benzalkonium chloride)Biguanidines (e.g. chlorhexidine)

Dyes (e.g. acriflavine)

SdrM MFS n.i. Hydrophilic fluoroquinolones (e.g. norfloxacin)Dyes (e.g. ethidium bromide, acriflavine) [42]
LmrS MFS n.i. Oxazolidinone (linezolid)Phenicols (e.g. choramphenicol, florfenicol)

Trimethoprim, erythromycin, kanamycin, fusidic acid

QACs (e.g. tetraphenylphosphonium)

Detergents (e.g. sodium docecyl sulphate)

Dyes (e.g. ethidium bromide)


Plasmid-encoded Efflux Systems

QacA MFS QacR QACs (e.g. tetraphenylphosphonium, benzalkonium chloride, dequalinium)Biguanidines (e.g. chlorhexidine)

Diamidines (e.g. pentamidine)

Dyes (e.g. ethidium bromide, rhodamine, acriflavine)

QacB MFS QacR QACs (e.g. tetraphenylphosphonium, benzalkonium chloride)Dyes (e.g. ethidium bromide, rhodamine, acriflavine) [53]
Smr SMR n.i. QACs (e.g. benzalkonium chloride, cetrimide)Dyes (e.g. ethidium bromide) [58,61]
QacG SMR n.i. QACs (e.g. benzalkonium chloride, cetyltrymethylammonium)Dyes (e.g. ethidium bromide) [67]
QacH SMR n.i. QACs (e.g. benzalkonium chloride, cetyltrymethylammonium)Dyes (e.g. ethidium bromide) [68]
QacJ SMR n.i. QACs (e.g. benzalkonium chloride, cetyltrymethylammonium)Dyes (e.g. ethidium bromide) [69]

a n.d.: The family of transporters to which SepA belongs is not elucidated to date.
b n.i.: The transporter has no regulator identified to date.
QACs: quaternary ammonium compounds
The importance of efflux pumps in bacterial antibiotic resistance

  1. A. Webber and L. J. V. Piddock
    Journal of Antimicrobial Chemotherapy (2003) 51, 9–11 pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics) from within cells into the external environment. These proteins are found in both Gram-positive and -negative bacteria as well as in eukaryotic organisms. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug resistance (MDR). In the prokaryotic kingdom there are five major families of efflux transporter: MF (major facilitator), MATE (multidrug and toxic efflux), RND (resistance-nodulation-division), SMR (small multidrug resistance) and ABC (ATP binding cassette). All these systems utilize the proton motive force as an energy source. Advances in DNA technology have led to the identification of members of the above families. Transporters that efflux multiple substrates, including antibiotics, have not evolved in response to the stresses of the antibiotic era. All bacterial genomes studied contain efflux pumps that indicate their ancestral origins. It has been estimated that ∼5–10% of all bacterial genes are involved in transport and a large proportion of these encode efflux pumps.
The efflux pump

The efflux pump

Multidrug-resistance efflux pumps — not just for resistance

Laura J. V. Piddock
Nature Reviews | Microbiology | Aug 2006; 4: 629

It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host defense molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.


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Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014

Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.

While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.

The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.

As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.

Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.  ….

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.

Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits.  …

The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

Gut Microbiome Heritability

Analyzing data from a large twin study, researchers have homed in on how host genetics can shape the gut microbiome.
By Tracy Vence | The Scientist Nov 6, 2014

Previous research suggested host genetic variation can influence microbial phenotype, but an analysis of data from a large twin study published in Cell today (November 6) solidifies the connection between human genotype and the composition of the gut microbiome. Studying more than 1,000 fecal samples from 416 monozygotic and dizygotic twin pairs, Cornell University’s Ruth Ley and her colleagues have homed in on one bacterial taxon, the family Christensenellaceae, as the most highly heritable group of microbes in the human gut. The researchers also found that Christensenellaceae—which was first described just two years ago—is central to a network of co-occurring heritable microbes that is associated with lean body mass index (BMI).  …

Of particular interest was the family Christensenellaceae, which was the most heritable taxon among those identified in the team’s analysis of fecal samples obtained from the TwinsUK study population.

While microbiologists had previously detected 16S rRNA sequences belonging to Christensenellaceae in the human microbiome, the family wasn’t named until 2012. “People hadn’t looked into it, partly because it didn’t have a name . . . it sort of flew under the radar,” said Ley.

Ley and her colleagues discovered that Christensenellaceae appears to be the hub in a network of co-occurring heritable taxa, which—among TwinsUK participants—was associated with low BMI. The researchers also found that Christensenellaceae had been found at greater abundance in low-BMI twins in older studies.

To interrogate the effects of Christensenellaceae on host metabolic phenotype, the Ley’s team introduced lean and obese human fecal samples into germ-free mice. They found animals that received lean fecal samples containing more Christensenellaceae showed reduced weight gain compared with their counterparts. And treatment of mice that had obesity-associated microbiomes with one member of the Christensenellaceae family, Christensenella minuta, led to reduced weight gain.   …

Ley and her colleagues are now focusing on the host alleles underlying the heritability of the gut microbiome. “We’re running a genome-wide association analysis to try to find genes—particular variants of genes—that might associate with higher levels of these highly heritable microbiota.  . . . Hopefully that will point us to possible reasons they’re heritable,” she said. “The genes will guide us toward understanding how these relationships are maintained between host genotype and microbiome composition.”

J.K. Goodrich et al., “Human genetics shape the gut microbiome,” Cell,, 2014.

Light-Operated Drugs

Scientists create a photosensitive pharmaceutical to target a glutamate receptor.
By Ruth Williams | The Scentist Nov 1, 2014

light operated drugs MO1

light operated drugs MO1

The desire for temporal and spatial control of medications to minimize side effects and maximize benefits has inspired the development of light-controllable drugs, or optopharmacology. Early versions of such drugs have manipulated ion channels or protein-protein interactions, “but never, to my knowledge, G protein–coupled receptors [GPCRs], which are one of the most important pharmacological targets,” says Pau Gorostiza of the Institute for Bioengineering of Catalonia, in Barcelona.

Gorostiza has taken the first step toward filling that gap, creating a photosensitive inhibitor of the metabotropic glutamate 5 (mGlu5) receptor—a GPCR expressed in neurons and implicated in a number of neurological and psychiatric disorders. The new mGlu5 inhibitor—called alloswitch-1—is based on a known mGlu receptor inhibitor, but the simple addition of a light-responsive appendage, as had been done for other photosensitive drugs, wasn’t an option. The binding site on mGlu5 is “extremely tight,” explains Gorostiza, and would not accommodate a differently shaped molecule. Instead, alloswitch-1 has an intrinsic light-responsive element.

In a human cell line, the drug was active under dim light conditions, switched off by exposure to violet light, and switched back on by green light. When Gorostiza’s team administered alloswitch-1 to tadpoles, switching between violet and green light made the animals stop and start swimming, respectively.

The fact that alloswitch-1 is constitutively active and switched off by light is not ideal, says Gorostiza. “If you are thinking of therapy, then in principle you would prefer the opposite,” an “on” switch. Indeed, tweaks are required before alloswitch-1 could be a useful drug or research tool, says Stefan Herlitze, who studies ion channels at Ruhr-Universität Bochum in Germany. But, he adds, “as a proof of principle it is great.” (Nat Chem Biol,, 2014)

Enhanced Enhancers

The recent discovery of super-enhancers may offer new drug targets for a range of diseases.
By Eric Olson | The Scientist Nov 1, 2014

To understand disease processes, scientists often focus on unraveling how gene expression in disease-associated cells is altered. Increases or decreases in transcription—as dictated by a regulatory stretch of DNA called an enhancer, which serves as a binding site for transcription factors and associated proteins—can produce an aberrant composition of proteins, metabolites, and signaling molecules that drives pathologic states. Identifying the root causes of these changes may lead to new therapeutic approaches for many different diseases.

Although few therapies for human diseases aim to alter gene expression, the outstanding examples—including antiestrogens for hormone-positive breast cancer, antiandrogens for prostate cancer, and PPAR-γ agonists for type 2 diabetes—demonstrate the benefits that can be achieved through targeting gene-control mechanisms.  Now, thanks to recent papers from laboratories at MIT, Harvard, and the National Institutes of Health, researchers have a new, much bigger transcriptional target: large DNA regions known as super-enhancers or stretch-enhancers. Already, work on super-enhancers is providing insights into how gene-expression programs are established and maintained, and how they may go awry in disease.  Such research promises to open new avenues for discovering medicines for diseases where novel approaches are sorely needed.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions (Cell, 153:307-19, 2013). They also appear to bind a large percentage of the transcriptional machinery compared to typical enhancers, allowing them to better establish and enforce cell-type specific transcriptional programs (Cell, 153:320-34, 2013).

Super-enhancers are closely associated with genes that dictate cell identity, including those for cell-type–specific master regulatory transcription factors. This observation led to the intriguing hypothesis that cells with a pathologic identity, such as cancer cells, have an altered gene expression program driven by the loss, gain, or altered function of super-enhancers.

Sure enough, by mapping the genome-wide location of super-enhancers in several cancer cell lines and from patients’ tumor cells, we and others have demonstrated that genes located near super-enhancers are involved in processes that underlie tumorigenesis, such as cell proliferation, signaling, and apoptosis.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions.

Genome-wide association studies (GWAS) have found that disease- and trait-associated genetic variants often occur in greater numbers in super-enhancers (compared to typical enhancers) in cell types involved in the disease or trait of interest (Cell, 155:934-47, 2013). For example, an enrichment of fasting glucose–associated single nucleotide polymorphisms (SNPs) was found in the stretch-enhancers of pancreatic islet cells (PNAS, 110:17921-26, 2013). Given that some 90 percent of reported disease-associated SNPs are located in noncoding regions, super-enhancer maps may be extremely valuable in assigning functional significance to GWAS variants and identifying target pathways.

Because only 1 to 2 percent of active genes are physically linked to a super-enhancer, mapping the locations of super-enhancers can be used to pinpoint the small number of genes that may drive the biology of that cell. Differential super-enhancer maps that compare normal cells to diseased cells can be used to unravel the gene-control circuitry and identify new molecular targets, in much the same way that somatic mutations in tumor cells can point to oncogenic drivers in cancer. This approach is especially attractive in diseases for which an incomplete understanding of the pathogenic mechanisms has been a barrier to discovering effective new therapies.

Another therapeutic approach could be to disrupt the formation or function of super-enhancers by interfering with their associated protein components. This strategy could make it possible to downregulate multiple disease-associated genes through a single molecular intervention. A group of Boston-area researchers recently published support for this concept when they described inhibited expression of cancer-specific genes, leading to a decrease in cancer cell growth, by using a small molecule inhibitor to knock down a super-enhancer component called BRD4 (Cancer Cell, 24:777-90, 2013).  More recently, another group showed that expression of the RUNX1 transcription factor, involved in a form of T-cell leukemia, can be diminished by treating cells with an inhibitor of a transcriptional kinase that is present at the RUNX1 super-enhancer (Nature, 511:616-20, 2014).

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization 
Andrea Sánchez-Vallet, et al.   eLife 2013;2:e00790

LysM effector

LysM effector

While host immune receptors

  • detect pathogen-associated molecular patterns to activate immunity,
  • pathogens attempt to deregulate host immunity through secreted effectors.

Fungi employ LysM effectors to prevent

  • recognition of cell wall-derived chitin by host immune receptors

Structural analysis of the LysM effector Ecp6 of

  • the fungal tomato pathogen Cladosporium fulvum reveals
  • a novel mechanism for chitin binding,
  • mediated by intrachain LysM dimerization,

leading to a chitin-binding groove that is deeply buried in the effector protein.

This composite binding site involves

  • two of the three LysMs of Ecp6 and
  • mediates chitin binding with ultra-high (pM) affinity.

The remaining singular LysM domain of Ecp6 binds chitin with

  • low micromolar affinity but can nevertheless still perturb chitin-triggered immunity.

Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.

Mutated Genes in Schizophrenia Map to Brain Networks
From –  Sep 3, 2013

Previous studies have shown that many people with schizophrenia have de novo, or new, genetic mutations. These misspellings in a gene’s DNA sequence

  • occur spontaneously and so aren’t shared by their close relatives.

Dr. Mary-Claire King of the University of Washington in Seattle and colleagues set out to

  • identify spontaneous genetic mutations in people with schizophrenia and
  • to assess where and when in the brain these misspelled genes are turned on, or expressed.

The study was funded in part by NIH’s National Institute of Mental Health (NIMH). The results were published in the August 1, 2013, issue of Cell.

The researchers sequenced the exomes (protein-coding DNA regions) of 399 people—105 with schizophrenia plus their unaffected parents and siblings. Gene variations
that were found in a person with schizophrenia but not in either parent were considered spontaneous.

The likelihood of having a spontaneous mutation was associated with

  • the age of the father in both affected and unaffected siblings.

Significantly more mutations were found in people

  • whose fathers were 33-45 years at the time of conception compared to 19-28 years.

Among people with schizophrenia, the scientists identified

  • 54 genes with spontaneous mutations
  • predicted to cause damage to the function of the protein they encode.

The researchers used newly available database resources that show

  • where in the brain and when during development genes are expressed.

The genes form an interconnected expression network with many more connections than

  • that of the genes with spontaneous damaging mutations in unaffected siblings.

The spontaneously mutated genes in people with schizophrenia

  • were expressed in the prefrontal cortex, a region in the front of the brain.

The genes are known to be involved in important pathways in brain development. Fifty of these genes were active

  • mainly during the period of fetal development.

“Processes critical for the brain’s development can be revealed by the mutations that disrupt them,” King says. “Mutations can lead to loss of integrity of a whole pathway,
not just of a single gene.”

These findings support the concept that schizophrenia may result, in part, from

  • disruptions in development in the prefrontal cortex during fetal development.

James E. Darnell’s “Reflections”

A brief history of the discovery of RNA and its role in transcription — peppered with career advice
By Joseph P. Tiano

James Darnell begins his Journal of Biological Chemistry “Reflections” article by saying, “graduate students these days

  • have to swim in a sea virtually turgid with the daily avalanche of new information and
  • may be momentarily too overwhelmed to listen to the aging.

I firmly believe how we learned what we know can provide useful guidance for how and what a newcomer will learn.” Considering his remarkable discoveries in

  • RNA processing and eukaryotic transcriptional regulation

spanning 60 years of research, Darnell’s advice should be cherished. In his second year at medical school at Washington University School of Medicine in St. Louis, while
studying streptococcal disease in Robert J. Glaser’s laboratory, Darnell realized he “loved doing the experiments” and had his first “career advancement event.”
He and technician Barbara Pesch discovered that in vivo penicillin treatment killed streptococci only in the exponential growth phase and not in the stationary phase. These
results were published in the Journal of Clinical Investigation and earned Darnell an interview with Harry Eagle at the National Institutes of Health.

Darnell arrived at the NIH in 1956, shortly after Eagle  shifted his research interest to developing his minimal essential cell culture medium, still used. Eagle, then studying cell metabolism, suggested that Darnell take up a side project on poliovirus replication in mammalian cells in collaboration with Robert I. DeMars. DeMars’ Ph.D.
adviser was also James  Watson’s mentor, so Darnell met Watson, who invited him to give a talk at Harvard University, which led to an assistant professor position
at the MIT under Salvador Luria. A take-home message is to embrace side projects, because you never know where they may lead: this project helped to shape
his career.

Darnell arrived in Boston in 1961. Following the discovery of DNA’s structure in 1953, the world of molecular biology was turning to RNA in an effort to understand how
proteins are made. Darnell’s background in virology (it was discovered in 1960 that viruses used RNA to replicate) was ideal for the aim of his first independent lab:
exploring mRNA in animal cells grown in culture. While at MIT, he developed a new technique for purifying RNA along with making other observations

  • suggesting that nonribosomal cytoplasmic RNA may be involved in protein synthesis.

When Darnell moved to Albert Einstein College of Medicine for full professorship in 1964,  it was hypothesized that heterogenous nuclear RNA was a precursor to mRNA.
At Einstein, Darnell discovered RNA processing of pre-tRNAs and demonstrated for the first time

  • that a specific nuclear RNA could represent a possible specific mRNA precursor.

In 1967 Darnell took a position at Columbia University, and it was there that he discovered (simultaneously with two other labs) that

  • mRNA contained a polyadenosine tail.

The three groups all published their results together in the Proceedings of the National Academy of Sciences in 1971. Shortly afterward, Darnell made his final career move
four short miles down the street to Rockefeller University in 1974.

Over the next 35-plus years at Rockefeller, Darnell never strayed from his original research question: How do mammalian cells make and control the making of different
mRNAs? His work was instrumental in the collaborative discovery of

  • splicing in the late 1970s and
  • in identifying and cloning many transcriptional activators.

Perhaps his greatest contribution during this time, with the help of Ernest Knight, was

  • the discovery and cloning of the signal transducers and activators of transcription (STAT) proteins.

And with George Stark, Andy Wilks and John Krowlewski, he described

  • cytokine signaling via the JAK-STAT pathway.

Darnell closes his “Reflections” with perhaps his best advice: Do not get too wrapped up in your own work, because “we are all needed and we are all in this together.”

Darnell Reflections - James_Darnell

Darnell Reflections – James_Darnell

Recent findings on presenilins and signal peptide peptidase

By Dinu-Valantin Bălănescu

γ-secretase and SPP

γ-secretase and SPP

Fig. 1 from the minireview shows a schematic depiction of γ-secretase and SPP

GxGD proteases are a family of intramembranous enzymes capable of hydrolyzing

  • the transmembrane domain of some integral membrane proteins.

The GxGD family is one of the three families of

  • intramembrane-cleaving proteases discovered so far (along with the rhomboid and site-2 protease) and
  • includes the γ-secretase and the signal peptide peptidase.

Although only recently discovered, a number of functions in human pathology and in numerous other biological processes

  • have been attributed to γ-secretase and SPP.

Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo highlighted the latest findings on the structure and function of γ-secretase and SPP
in a recent minireview in The Journal of Biological Chemistry.

  • γ-secretase is involved in cleaving the amyloid-β precursor protein, thus producing amyloid-β peptide,

the main component of senile plaques in Alzheimer’s disease patients’ brains. The complete structure of mammalian γ-secretase is not yet known; however,
Tomita and Iwatsubo note that biochemical analyses have revealed it to be a multisubunit protein complex.

  • Its catalytic subunit is presenilin, an aspartyl protease.

In vitro and in vivo functional and chemical biology analyses have revealed that

  • presenilin is a modulator and mandatory component of the γ-secretase–mediated cleavage of APP.

Genetic studies have identified three other components required for γ-secretase activity:

  1. nicastrin,
  2. anterior pharynx defective 1 and
  3. presenilin enhancer 2.

By coexpression of presenilin with the other three components, the authors managed to

  • reconstitute γ-secretase activity.

Tomita and Iwatsubo determined using the substituted cysteine accessibility method and by topological analyses, that

  • the catalytic aspartates are located at the center of the nine transmembrane domains of presenilin,
  • by revealing the exact location of the enzyme’s catalytic site.

The minireview also describes in detail the formerly enigmatic mechanism of γ-secretase mediated cleavage.

SPP, an enzyme that cleaves remnant signal peptides in the membrane

  • during the biogenesis of membrane proteins and
  • signal peptides from major histocompatibility complex type I,
  • also is involved in the maturation of proteins of the hepatitis C virus and GB virus B.

Bioinformatics methods have revealed in fruit flies and mammals four SPP-like proteins,

  • two of which are involved in immunological processes.

By using γ-secretase inhibitors and modulators, it has been confirmed

  • that SPP shares a similar GxGD active site and proteolytic activity with γ-secretase.

Upon purification of the human SPP protein with the baculovirus/Sf9 cell system,

  • single-particle analysis revealed further structural and functional details.

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection

From –  Sep 3, 2013 4:24 PM

Experimental and computational evidence suggests that

  • HLAs preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that
  • this preference may provide improved clearance of infection in several viral systems.

To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study
performed during the 2009–2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with

  • IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression).

A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,

  • were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
  • are common in certain indigenous populations in which increased pH1N1 morbidity has been reported.

HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection.
The computational tools used in this study may be useful predictors of potential morbidity and

  • identify immunologic differences of new variant influenza strains
  • more accurately than evolutionary sequence comparisons.

Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases

  • might advance clinical practices for severe H1N1 infections among genetically susceptible populations.

Metabolomics in drug target discovery

J D Rabinowitz et al.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ.
Cold Spring Harbor Symposia on Quantitative Biology 11/2011; 76:235-46. 

Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes,

  • metabolomics can point to potential new sites for therapeutic intervention.

Particularly promising enzymatic targets are those that

  • carry increased flux in the disease state.

Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for

  • finding new drug targets using metabolomics and isotope tracers.

The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus,

  • metabolomic analysis of infected versus mock-infected cells revealed
  • dramatic concentration changes around the current antiviral target enzymes.

Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes

  • in a region of metabolism unrelated to the current antiviral target.

Instead, it pointed to the tricarboxylic acid (TCA) cycle and

  • its efflux to feed fatty acid biosynthesis as a potential preferred target.

Isotope tracer studies revealed that cytomegalovirus greatly increases flux through

  • the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase.
  • Inhibition of this enzyme blocks human cytomegalovirus replication.

Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of

  • metabolomics as a drug target discovery strategy will be
  • successful clinical development of therapeutics hitting these new targets.

 Related References

Use of metabolic pathway flux information in targeted cancer drug design. Drug Discovery Today: Therapeutic Strategies 1:435-443, 2004.

Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics. 2005;5(5):293-302. Review. PMID: 16196499

Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.
Mini Rev Med Chem.  2005 Jan;5(1):13-20. Review. PMID: 15638788 [PubMed – indexed for MEDLINE] Related citations

Development of Tracer-Based Metabolomics and its Implications for the Pharmaceutical Industry. Int J Pharm Med 2007; 21 (3): 217-224.

Use of metabolic pathway flux information in anticancer drug design. Ernst Schering Found Symp Proc. 2007;(4):189-203. Review. PMID: 18811058

Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice. J Pharmacol Exp Ther. 2011 Jul;338(1):70-81. PMID: 21471191

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the
[U-C(6)]-d-glucose tracer in mice. Metabolomics. 2009 Sep;5(3):336-345. PMID: 19718458

Metabolic Pathways as Targets for Drug Screening, Metabolomics, Dr Ute Roessner (Ed.), ISBN: 978-953-51-0046-1, InTech, Available from:

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. FASEB J. 2013 Jul;27(7):2845-54. PMID: 23515442

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Drug Discov. Today 19 (2014), 171–182


  • We now have metabolic network models; the metabolome is represented by their nodes.
  • Metabolite levels are sensitive to changes in enzyme activities.
  • Drugs hitchhike on metabolite transporters to get into and out of cells.
  • The consensus network Recon2 represents the present state of the art, and has predictive power.
  • Constraint-based modelling relates network structure to metabolic fluxes.

Metabolism represents the ‘sharp end’ of systems biology, because changes in metabolite concentrations are

  • necessarily amplified relative to changes in the transcriptome, proteome and enzyme activities, which can be modulated by drugs.

To understand such behaviour, we therefore need (and increasingly have) reliable consensus (community) models of

  • the human metabolic network that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters, because

  • drugs bear structural similarities to metabolites known from the network reconstructions and
  • from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic network reconstruction; it can predict inter alia:

(i) the effects of inborn errors of metabolism;

(ii) which metabolites are exometabolites, and

(iii) how metabolism varies between tissues and cellular compartments.

However, even these qualitative network models are not yet complete. As our understanding improves

  • so do we recognise more clearly the need for a systems (poly)pharmacology.

Introduction – a systems biology approach to drug discovery

It is clearly not news that the productivity of the pharmaceutical industry has declined significantly during recent years

  • following an ‘inverse Moore’s Law’, Eroom’s Law, or
  • that many commentators, consider that the main cause of this is
  • because of an excessive focus on individual molecular target discovery rather than a more sensible strategy
  • based on a systems-level approach (Fig. 1).
drug discovery science

drug discovery science

Figure 1.

The change in drug discovery strategy from ‘classical’ function-first approaches (in which the assay of drug function was at the tissue or organism level),
with mechanistic studies potentially coming later, to more-recent target-based approaches where initial assays usually involve assessing the interactions
of drugs with specified (and often cloned, recombinant) proteins in vitro. In the latter cases, effects in vivo are assessed later, with concomitantly high levels of attrition.

Arguably the two chief hallmarks of the systems biology approach are:

(i) that we seek to make mathematical models of our systems iteratively or in parallel with well-designed ‘wet’ experiments, and
(ii) that we do not necessarily start with a hypothesis but measure as many things as possible (the ’omes) and

  • let the data tell us the hypothesis that best fits and describes them.

Although metabolism was once seen as something of a Cinderella subject,

  • there are fundamental reasons to do with the organisation of biochemical networks as
  • to why the metabol(om)ic level – now in fact seen as the ‘apogee’ of the ’omics trilogy –
  •  is indeed likely to be far more discriminating than are
  • changes in the transcriptome or proteome.

The next two subsections deal with these points and Fig. 2 summarises the paper in the form of a Mind Map.

metabolomics and systems pharmacology

metabolomics and systems pharmacology

Metabolic Disease Drug Discovery— “Hitting the Target” Is Easier Said Than Done

David E. Moller, et al.

Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated. Continuing

  • unmet medical needs remain a major driver for new research.

Drug discovery approaches in this field have mirrored industry trends, leading to a recent

  • increase in the number of molecules entering development.

However, worrisome trends and newer hurdles are also apparent. The history of two newer drug classes—

  1. glucagon-like peptide-1 receptor agonists and
  2. dipeptidyl peptidase-4 inhibitors—

illustrates both progress and challenges. Future success requires that researchers learn from these experiences and

  • continue to explore and apply new technology platforms and research paradigms.

The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts an even greater diabetes burden (>430 million people afflicted) by 2030, which will disproportionately affect developing nations (International Diabetes Federation, 2011). Yet

  • existing drug classes for diabetes, obesity, and comorbid cardiovascular (CV) conditions have substantial limitations.

Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable shortcomings. In general,

Therefore, clinicians must often use combination therapy, adding additional agents over time. Ultimately many patients will need to use insulin—a therapeutic class first introduced in 1922. Most existing agents also have

  • issues around safety and tolerability as well as dosing convenience (which can impact patient compliance).

Pharmacometabolomics, also known as pharmacometabonomics, is a field which stems from metabolomics,

  • the quantification and analysis of metabolites produced by the body.

It refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to

  • predict or evaluate the metabolism of pharmaceutical compounds, and
  • to better understand the pharmacokinetic profile of a drug.

Alternatively, pharmacometabolomics can be applied to measure metabolite levels

  • following the administration of a pharmaceutical compound, in order to
  • monitor the effects of the compound on certain metabolic pathways(pharmacodynamics).

This provides detailed mapping of drug effects on metabolism and

  • the pathways that are implicated in mechanism of variation of response to treatment.

In addition, the metabolic profile of an individual at baseline (metabotype) provides information about

  • how individuals respond to treatment and highlights heterogeneity within a disease state.

All three approaches require the quantification of metabolites found

relationship between -OMICS

relationship between -OMICS

Pharmacometabolomics is thought to provide information that

Looking at the characteristics of an individual down through these different levels of detail, there is an

  • increasingly more accurate prediction of a person’s ability to respond to a pharmaceutical compound.
  1. the genome, made up of 25 000 genes, can indicate possible errors in drug metabolism;
  2. the transcriptome, made up of 85,000 transcripts, can provide information about which genes important in metabolism are being actively transcribed;
  3. and the proteome, >10,000,000 members, depicts which proteins are active in the body to carry out these functions.

Pharmacometabolomics complements the omics with

  • direct measurement of the products of all of these reactions, but with perhaps a relatively
  • smaller number of members: that was initially projected to be approximately 2200 metabolites,

but could be a larger number when gut derived metabolites and xenobiotics are added to the list. Overall, the goal of pharmacometabolomics is

  • to more closely predict or assess the response of an individual to a pharmaceutical compound,
  • permitting continued treatment with the right drug or dosage
  • depending on the variations in their metabolism and ability to respond to treatment.

Pharmacometabolomic analyses, through the use of a metabolomics approach,

  • can provide a comprehensive and detailed metabolic profile or “metabolic fingerprint” for an individual patient.

Such metabolic profiles can provide a complete overview of individual metabolite or pathway alterations,

This approach can then be applied to the prediction of response to a pharmaceutical compound

  • by patients with a particular metabolic profile.

Pharmacometabolomic analyses of drug response are

Pharmacogenetics focuses on the identification of genetic variations (e.g. single-nucleotide polymorphisms)

  • within patients that may contribute to altered drug responses and overall outcome of a certain treatment.

The results of pharmacometabolomics analyses can act to “inform” or “direct”

  • pharmacogenetic analyses by correlating aberrant metabolite concentrations or metabolic pathways to potential alterations at the genetic level.

This concept has been established with two seminal publications from studies of antidepressants serotonin reuptake inhibitors

  • where metabolic signatures were able to define a pathway implicated in response to the antidepressant and
  • that lead to identification of genetic variants within a key gene
  • within the highlighted pathway as being implicated in variation in response.

These genetic variants were not identified through genetic analysis alone and hence

  • illustrated how metabolomics can guide and inform genetic data.

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Andrea Trochine, Darren J. Creek, Paula Faral-Tello, Michael P. Barrett, Carlos Robello
Published: May 22, 2014

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi,

  • involves administration of benznidazole (Bzn).

Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active. We used a

  • non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols

  1. trypanothione,
  2. homotrypanothione and
  3. cysteine

were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment.

These metabolites included reduction products, fragments and covalent adducts of reduced Bzn

  • linked to each of the major low molecular weight thiols:
  1. trypanothione,
  2. glutathione,
  3. g-glutamylcysteine,
  4. glutathionylspermidine,
  5. cysteine and
  6. ovothiol A.

Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI,

  • were found within the parasites,
  • but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Our data is indicative of a major role of the

  • thiol binding capacity of Bzn reduction products
  • in the mechanism of Bzn toxicity against T. cruzi.



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Introduction to Proteomics

Author and Curator: Larry H. Bernstein, MD, FCAP  


We have had a considerable extended discussion of preoteins and peptides, protein sinthesis, amino acid incorporation into protein, and metabolism of carbohydrates and lipids.  It is also clear that the historic practice of medicine, and the classification of biological systems has been highly dependent on the observations related to the observed phenotypical traits and disturbances of normal function that could be measured by traditional metabolic pathways for over a century.

What did we gain from the genomic revolution?

  1. Traceability of protein expression to a basic coded message
  2. The possibility of tracing disturbed cellular function to mutation related loss-of-function
  3. The ability to trace generational traits over long periods of time
  4. The promise of regenerating the enterprise of pharmacology and pharmaceutical intervention based on the silencing of or readjustment of regulated metabolic pathways to bring an adaptive rebalancing favoring extended life

What can we expect as we progress further as a result of the last two decades?

  1. There is a huge amount of information, as well as missing information that is necessary for adequately tackling the mastery of the life processes.
  2. There is a complex web of knowledge that goes beyond the genome and the one-gene one-enzyme, and the DNA-RNA-protein hypotheses that can only be realized by more full disclosure of the many metabolic control circuits involved in cellular homeostasis and adaptive control.
  3. The ability to come to disclosure and understanding of this cellular balancing will require the comprehensive exploration of the proteome and the active role of proteins and peptides in the functioning of all cells, and the organism.
  4. Proteomics will open up the discovery of new approaches to diagnostics and pharmaceutical discovery.

What about proteins?  What can proteins do? What can’t they do!

  • Enzymes are proteins that make sure that chemical reactions in your body take place up to a million times faster than they would without enzymes.
  • Antibodies are proteins that help your immune system to fight disease.
  • When you get an injury, the bleeding stops because of blood clots, thanks to the proteins fibrinogen and thrombin.
  • Transport! Some proteins carry vitamins ot hormones from one place to another, or form tunnels (pores) in cell membranes that will let only specific molecules (or ions) through. Hemoglobin, a protein in your blood, carries oxygen from your lungs to your cells.
  • Strength and support! Other proteins like collagen and keratin are strong and tough and make up your skin, hair, and fingernails. Collagen also supports your cells and organs so they don’t slosh around.
  • Motion! The proteins myosin and actin make up much of your muscle tissue. They work together so your muscles can move you around. Some bacteria have cilia and flagella made out of proteins. The bacteria can whip these around to move from place to place.

Proteins (/ˈprˌtnz/ or /ˈprti.ɨnz/) are large biological molecules, or macromolecules,

Proteins perform a vast array of functions within living organisms, including

  1. catalyzing metabolic reactions,
  2. replicating DNA,
  3. responding to stimuli, and
  4. transporting molecules from one location to another.

Proteins differ from one another primarily in

  1. their sequence of amino acids,
  2. which is dictated by the nucleotide sequence of their genes, and
  3. which usually results in folding of the protein into

A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than about 20-30 residues, are rarely considered to be proteins and are commonly called peptides, or sometimes oligopeptides. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by

In general, the genetic code specifies 20 standard amino acids; however, in certain organisms the genetic code can include selenocysteine and—in certain archaeapyrrolysine. Shortly after or even during synthesis,

  • the residues in a protein are often chemically modified by posttranslational modification,
  • which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins.

Posttranslational modification (PTM) is a step in protein biosynthesis. Proteins created by ribosomes translating mRNA into polypeptide chains may undergo PTM (such as folding, cutting and other processes) before becoming the mature protein product.  After translation, the posttranslational modification of amino acids extends the range of functions of the protein by attaching it to other biochemical functional groups (such as acetate, phosphate, various lipids and carbohydrates), changing the chemical nature of an amino acid (e.g. citrullination), or making structural changes (e.g. formation of disulfide bridges).

Also, enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. Also, most nascent polypeptides start with the amino acid methionine because the “start” n mRNA also codes for this amino acid. This amino acid is usually taken off during post-translational modification. Other modifications, like phosphorylation, are part of common mechanisms for controlling the behavior of a protein, for instance activating or inactivating an enzyme.

posttranslational modification of insulin

posttranslational modification of insulin

Posttranslational modification of insulin. At the top, the ribosome translates a mRNA sequence into a protein, insulin, and passes the protein through the endoplasmic reticulum, where it is cut, folded and held in shape by disulfide (-S-S-) bonds. Then the protein passes through the golgi apparatus, where it is packaged into a vesicle. In the vesicle, more parts are cut off, and it turns into mature insulin.

Genetic Code mapped

Genetic Code mapped

The genetic code diagram showing the amino acid residues as target of modification.

PTMs involving addition of cofactors for enhanced enzymatic activity

Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors.  Examples of cofactors include metal ions like iron and zinc. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes.



Coenzymes are molecules that work at the active site of an enzyme and aid in recognizing, attracting, or repulsing a substrate or product. Many are derived from vitamins. The substrate is the molecule upon which an enzyme catalyzes a reaction transforming A to B by removal or addition of a hydrogen, or a hydroxyl group, or a methyl group, and so forth. This is  how an alcohol or an aldehyde is produced. Such a reaction is critical is carbohydrate metabolism for producing two 3-carbon sugars from a 6-carbon sugar. Coenzymes shuttle chemical groups from one enzyme to another enzyme. They may bind loosely to enzymes, while another group of cofactors do not.

Prosthetic groups are cofactors that bind tightly to proteins or enzymes. As if holding on for dear life, they are not easily removed. They can be organic or metal ions and are often attached to proteins by a covalent bond. The same cofactors can bind multiple different types of enzymes and may bind some enzymes loosely, as a coenzyme, and others tightly, as a prosthetic group. Some cofactors may always tightly bind their enzymes. It’s important to note, though, that these prosthetic groups can also bind to proteins other than enzymes.  A holoenzyme is an enzyme with any metal ions or coenzymes attached to it that is now ready to catalyze a reaction.



Around the world, millions of people don’t get enough protein. Protein malnutrition leads to the condition known as kwashiorkor. Lack of protein can cause growth failure, loss of muscle mass, decreased immunity, weakening of the heart and respiratory system, and death.

All Protein Isn’t Alike

Protein is built from building blocks called amino acids. Our bodies make amino acids in two different ways: Either from scratch, or by modifying others. A few amino acids (known as the essential amino acids) must come from food.

  • Animal sources of protein tend to deliver all the amino acids we need.
  • Other protein sources, such as fruits, vegetables, grains, nuts and seeds, lack one or more essential amino acids.

Vegetarians need to be aware of this. People who don’t eat meat, fish, poultry, eggs, or dairy products need to eat a variety of protein-containing foods each day in order to get all the amino acids needed to make new protein.
Molecular Biologists Guide to Proteomics

PR. Graves and TA.J. Haystead*
Microbiol Mol Biol Rev. Mar 2002; 66(1): 39–63  PMC120780

The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that

  • the final product of a gene is inherently more complex and
  • closer to function than the gene itself.

Shortfalls in the ability of bioinformatics to predict

  • both the existence and function of genes have also illustrated
  • the need for protein analysis.

Moreover, only through the study of proteins can posttranslational modifications be determined,

  • which can profoundly affect protein function.

Proteomics has been enabled by

  • the accumulation of both DNA and protein sequence databases,
  • improvements in mass spectrometry, and
  • the development of computer algorithms for database searching.

In this review, we describe why proteomics is important,

  • how it is conducted, and
  • how it can be applied to complement other existing technologies.

We conclude that currently, the most practical application of proteomics is

  • the analysis of target proteins as opposed to entire proteomes.

This type of proteomics, referred to as functional proteomics, is always

  • driven by a specific biological question.

In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages

  • of a functional proteomics approach and

provide examples of how different methodologies can be utilized to address a wide variety of biological problems.

Entry of our laboratory into proteomics 5 years ago was driven by a need to define a complex mixture of proteins (∼36 proteins) we had affinity isolated that bound specifically to the catalytic subunit of protein phosphatase 1 (PP-1, a serine/threonine protein phosphatase that regulates multiple dephosphorylation events in cells). We were faced with the task of trying to understand the significance of these proteins, and the only obvious way to begin to do this was to identify them by sequencing. Since the majority of intact eukaryotic proteins are not immediately accessible to Edman sequencing

  • due to posttranslational N-terminal modifications,
  • we invented mixed-peptide sequencing.

This method enables internal peptide sequence information to be derived from proteins

  • electroblotted onto hydrophobic membranes.

Using the mixed-peptide sequencing strategy, we identified all 36 proteins in about a week. The mixture contained at least two known PP-1 regulatory subunits, but most were novel proteins of unknown function. Herein lies the lesson of proteomics. Identifying long lists of potentially interesting proteins often generates more questions than it seeks to answer.

Despite learning this obvious lesson, our early sequencing experiences were an epiphany that has subsequently altered our whole scientific strategy for probing protein function in cells. The sequencing of the 36 proteins has opened new avenues to further explore the functions of PP-1 in intact cells. Because of increased sensitivity, our approaches now routinely use state-of-the-art mass spectrometry (MS) techniques. However, rather than using proteomics to simply characterize large numbers of proteins in complex mixtures, we see the real application of this technology as a tool to enhance the power of existing approaches currently used by the modern molecular biologist such as classical yeast and mouse genetics, tissue culture, protein expression systems, and site-directed mutagenesis.

Importantly, the one message we would want the reader to take away from reading this review is that one should always let the biological question in mind drive the application of proteomics rather than simply engaging in an orgy of protein sequencing. From our experiences, we believe that if the appropriate controls are performed, proteomics is an extremely powerful approach for addressing important physiological questions. One should always design experiments to define a selected number of relevant proteins in the mixture of interest. Examples of such experiments that we routinely perform include defining early phosphorylation events in complex protein mixtures after hormone treatment of intact cells or comparing patterns of protein derived from a stimulated versus nonstimulated cell in an affinity pull-down experiment. Only the proteins that were specifically phosphorylated or bound in response to the stimulus are sequenced in the complex mixtures. Sequencing proteins that are regulated then has a meaningful outcome and directs all subsequent biological investigation.

The term “proteomics” was first coined in 1995 and was defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism. Today, two definitions of proteomics are encountered. The first is the more classical definition, restricting the large-scale analysis of gene products to studies involving only proteins. The second and more inclusive definition combines protein studies with analyses that have a genetic readout such as mRNA analysis, genomics, and the yeast two-hybrid analysis. However, the goal of proteomics remains the same, i.e., to obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually.

Using the more inclusive definition of proteomics, many different areas of study are now grouped under the rubric of proteomics (Fig. (Fig.1).1). These include protein-protein interaction studies, protein modifications, protein function, and protein localization studies to name a few. The aim of proteomics is not only to identify all the proteins in a cell but also to create a complete three-dimensional (3-D) map of the cell indicating where proteins are located. These ambitious goals will certainly require the involvement of a large number of different disciplines such as molecular biology, biochemistry, and bioinformatics. It is likely that in bioinformatics alone, more powerful computers will have to be devised to organize the immense amount of information generated from these endeavors.

Types of proteomics and their applications to biology

Types of proteomics and their applications to biology

In the quest to characterize the proteome of a given cell or organism, it should be remembered that the proteome is dynamic. The proteome of a cell will reflect the immediate environment in which it is studied. In response to internal or external cues, proteins can be modified by posttranslational modifications, undergo translocations within the cell, or be synthesized or degraded. Thus, examination of the proteome of a cell is like taking a “snapshot” of the protein environment at any given time. Considering all the possibilities, it is likely that any given genome can potentially give rise to an infinite number of proteomes.

The first major technology to emerge for the identification of proteins was the sequencing of proteins by Edman degradation. A major breakthrough was the development of microsequencing techniques for electroblotted proteins. This technique was used for the identification of proteins from 2-D gels to create the first 2-D databases.  One of the most important developments in protein identification has been the development of MS technology. In the last decade, the sensitivity of analysis and accuracy of results for protein identification by MS have increased by several orders of magnitude. It is now estimated that proteins in the femtomolar range can be identified in gels. Because MS is more sensitive, can tolerate protein mixtures, and is amenable to high-throughput operations, it has essentially replaced Edman sequencing as the protein identification tool of choice.

The growth of proteomics is a direct result of advances made in large-scale nucleotide sequencing of expressed sequence tags and genomic DNA. Without this information, proteins could not be identified even with the improvements made in MS. Protein identification (by MS or Edman sequencing) relies on the presence of some form of database for the given organism. The majority of DNA and protein sequence information has accumulated within the last 5 to 10 years. In 1995, the first complete genome of an organism was sequenced, that of Haemophilus influenzae. At the time of this writing, the sequencing of the genomes of 45 microorganisms has been completed and that of 170 more is under way ( To date, five eukaryotic genomes have been completed: Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, and Drosophila melanogaster. In addition, the rice, mouse, and human genomes are near completion.

One of the first applications of proteomics will be to identify the total number of genes in a given genome. This “functional annotation” of a genome is necessary because

  • it is still difficult to predict genes accurately from genomic data. One problem is that
  • the exon-intron structure of most genes cannot be accurately predicted by bioinformatics.

To achieve this goal, genomic information will have to be integrated with

  • data obtained from protein studies to confirm the existence of a particular gene.

The analysis of mRNA is

  • not a direct reflection of the protein content in the cell.

Many studies have shown a poor correlation

  • between mRNA and protein expression levels.

The formation of mRNA is only the first step in a long sequence of events resulting in the synthesis of a protein (Fig. (Fig.2).2).

  1. mRNA is subject to posttranscriptional control in the form of alternative splicing, polyadenylation, and mRNA editing. Many different protein isoforms can be generated from a single gene at this step.
  2. mRNA then can be subject to regulation at the level of protein translation. Proteins, having been formed, are subject to posttranslational modification. It is estimated that up to 200 different types of posttranslational protein modification exist. Proteins can also be regulated by proteolysis and compartmentalization. It is clear that the tenet of “one gene, one protein” is an oversimplification.
Mechanisms by which a single gene can give rise to multiple gene products

Mechanisms by which a single gene can give rise to multiple gene products

Mechanisms by which a single gene can give rise to multiple gene products. Multiple protein isoforms can be generated by RNA processing when RNA is alternatively spliced or edited to form mature mRNA. mRNA, in turn, can be regulated by stability and efficiency
One of the most important applications of proteomics will be the characterization of posttranslational protein modifications. Proteins are known to be modified posttranslationally in response to a variety of intracellular and extracellular signals. For example, protein phosphorylation is an important signaling mechanism and disregulation of protein kinases or phosphatases can result in oncogenesis. By using a proteomics approach, changes in the modifications of many proteins expressed by a cell can be analyzed simultaneously.
Of fundamental importance in biology is the understanding of protein-protein interactions. The process of cell growth, programmed cell death, and the decision to proceed through the cell cycle are all regulated by signal transduction through protein complexes. Proteomics aims to develop a complete 3-D map of all protein interactions in the cell. One step toward this goal was recently completed for the microorganism Helicobacter pylori. Using the yeast two-hybrid method to detect protein interactions, 1,200 connections were identified between H. pylori proteins covering 46.6% of the genome. A comprehensive two-hybrid analysis has also been performed on all the proteins from the yeast S. cerevisiae.
mixed peptide sequencing with MS

mixed peptide sequencing with MS

The process of mixed-peptide sequencing involves separation of a complex protein mixture by polyacrylamide gel electrophoresis (1-D or 2-D) and then transfer of the proteins to an inert membrane by electroblotting (Fig. (Fig.4).4). The proteins of interest are visualized on the membrane surface, excised, and fragmented chemically at methionine (by CNBr) or tryptophan (by skatole) into several large peptide fragments.
FASTF and FASTS search programs

FASTF and FASTS search programs

The mixed-sequence data are fed into the FASTF or TFASTF algorithms, which sort and match the data against protein (FASTF) and DNA (TFASTF) databases to unambiguously identify the protein. The FASTF and TFASTF programs were written in collaboration with William Pearson (Department of Biochemistry, University of Virginia). Because minimal sample handling is involved, mixed-peptide sequencing can be a sensitive approach for identifying proteins in polyacrylamide gels at the 0.1- to 1-pmol level.  A recent variation of T/FASTF has been devised for MS (101) (Fig. (Fig.5B).5B). The T/FASTF/S programs are available at (Table (Table11).

triple quadrupole MS

triple quadrupole MS

Triple-quadrupole mass spectrometers are most commonly used to obtain amino acid sequences. In the first stage of analysis, the machine is operated in MS scan mode and all ions above a certain m/z ratio are transmitted to the third quadrupole for mass analysis (Fig. (Fig.6)6) (82, 173). In the second stage, the mass spectrometer is operated in MS/MS mode and a particular peptide ion is selectively passed into the collision chamber. Inside the collision chamber, peptide ions are fragmented by interactions with an inert gas by a process known as collision-induced dissociation or collisionally activated dissociation. The peptide ion fragments are then resolved on the basis of their m/z ratio by the third quadrupole (Fig. (Fig.6).6). Since two different mass spectra are obtained in this analysis, it is referred to as tandem mass spectrometry (MS/MS). MS/MS is used to obtain the amino acid sequence of peptides by generating a series of peptides that differ in mass by a single amino acid.

The largest application of proteomics continues to be protein expression profiling. Through the use of two-dimensional gels or novel techniques such as ICAT, the expression levels of proteins or changes in their level of modification between two different samples can be compared and the proteins can be identified. This approach can facilitate the dissection of signaling mechanisms or identify disease-specific proteins.

Cancer cells are good candidates for proteomics studies because they can be compared to their non-transformed counterparts. Analysis of differentially expressed proteins in normal versus cancer cells can

(i) identify novel tumor cell biomarkers that can be used for diagnosis,

(ii) provide clues to mechanisms of cancer development, and

(iii) identify novel targets for therapeutic intervention. Protein expression profiling has been used in the study of breast, esophageal, bladder and prostate cancer. From these studies, tumor-specific proteins were identified and 2-D protein expression databases were generated. Many of these 2-D protein databases are now available on the World Wide Web.

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My Cancer Genome from Vanderbilt University: Matching Tumor Mutations to Therapies & Clinical Trials

Reporter: Aviva Lev-Ari, PhD, RN


GenomOncology Collaborates With Vanderbilt-Ingram Cancer Center for the Commercial Development of My Cancer Genome™ Tools

February 10, 2014

Westlake, OH. February 10, 2014 – GenomOncology and Vanderbilt-Ingram Cancer Center (VICC) today announced a partnership for the exclusive commercial development of a decision support tool based on My Cancer Genome™, an online precision cancer medicine knowledge resource for physicians, patients, caregivers and researchers.

Through this collaboration, GenomOncology and VICC will enhance My Cancer Genome through the development of a new genomics content management tool. The website will remain free and open to the public. In addition, GenomOncology will develop a decision support tool based on My Cancer Genome™ data that will enable automated interpretation of mutations in the genome of a patient’s tumor, providing actionable results in hours versus days. According to the terms of the agreement, all commercial use of My Cancer Genome™ in any form will be licensed through GenomOncology.

Vanderbilt-Ingram Cancer Center (VICC) launched My Cancer Genome™ in January 2011 as an integral part of their Personalized Cancer Medicine Initiative that helps physicians and researchers track the latest developments in precision cancer medicine and connect with clinical research trials. This web-based information tool is designed to quickly educate clinicians on the rapidly expanding list of genetic mutations that impact cancers and enable the research of treatment options based on specific mutations. For more information on My Cancer Genome™

“The future of cancer diagnostics and treatment is genomics-based precision medicine. Therapies based on the specific genetic alterations that underlie a patient’s cancer not only result in better outcomes but often have less adverse reactions,” commented Manuel Glynias, President and CEO of GenomOncology. “A resource like My Cancer Genome™ that matches tumor mutations to therapies and makes information accessible and convenient is an incredibly valuable tool. Our collaboration with Vanderbilt-Ingram Cancer Center on My Cancer Genome™ is designed to keep this resource comprehensive, scalable and easy for clinicians to use.”

“We are excited about the opportunity to collaborate with GenomOncology to enhance My Cancer Genome™ and develop tools for other hospitals, labs and cancer centers to use for patient care,” said Mia Levy M.D., Ph.D., co-founder of the My Cancer Genome™ site. “We believe in the need for high-quality, curated information to educate physicians and give them confidence as they make treatment decisions for patients.”

About Vanderbilt-Ingram Cancer Center

Vanderbilt-Ingram Cancer Center, a National Cancer Institute–designated Comprehensive Cancer Center, conducts basic, translational, and clinical research that offers adult and pediatric oncology treatment. VICC is a member of the National Comprehensive Cancer Network along with 22 other leading centers working together to improve quality and effectiveness of cancer care. For more information about VICC and precision medicine visit

About GenomOncology

GenomOncology is enabling precision medicine by translating next generation sequencing data into actionable information for clinicians and researchers. In collaboration with molecular pathologists and physicians, GenomOncology has developed the GO Clinical Workbench™, a decision support tool with a step-by-step workflow that takes raw data from the sequencer and translates the specific molecular profile of each patient’s tumor genome into an actionable clinical report. GenomOncology’s research platform, GenomAnalytics™, allows scientists to analyze one or hundreds of genomes simultaneously to look for causal variants, reducing the time required to understand the genomic alterations that lead to new discoveries about the biology of cancer. More information can be found on the company’s website at

For more information regarding this announcement, please contact:

Jane Krug, PR Contact
Phone: 504-390-5935




GenomOncology’s President and CEO


Mr. Glynias is a serial entrepreneur with over 25 years of experience in bioinformatics. Prior to GenomOncology, Manuel was a partner at Rosetta, a leading interactive marketing agency, where he helped develop big data solutions for ecommerce clients. In the late 90’s, Manuel was the founder and CEO of NetGenics, a venture-backed provider of discovery informatics to the biopharmaceutical industry and academic research centers. He also developed a number of other commercial software platforms including MacGene, Gene Works, and Primer Express. Manuel has an AB in Biochemistry and Molecular Biology from Harvard College.

GenomOncology’s BUSINESS MODEL

Our success depends on your success! The business model is comprised of two main components:

Up front fee

Nominal fee covers installation support, configuring the Workbench to your specification, designing and developing custom report(s) and training your team.

Per sample fee

GenomOncology is paid on signed-out clinical reports. This philosophy aligns GenomOncology with your Laboratory as we are incentivized to offer world-class support and solutions to differentiate your clinical NGS program. There is no annual license fee.

Optional Services

GenomOncology provides a variety of additional services including support of assay validation, integration with LIS / EMR, and custom new feature development.



GenomAnalytics THREE Video Demonstrations




What Is My Cancer Genome?

  • ​My Cancer Genome is a personalized cancer medicine knowledge resource for physicians, patients, caregivers and researchers.
  • My Cancer Genome gives up-to-date information on what mutations make cancers grow and related therapeutic implications, including available clinical trials.
  • My Cancer Genome is a one-stop tool that matches tumor mutations to therapies, making information accessible and convenient for busy clinicians.
  • For information about application programming interfaces (APIs) and licensing, please contact My Cancer Genome’s content licensee, GenomOncology, at or 440-617-6087.

Development Team

Mia Levy M.D., Ph.D., co-founder of the My Cancer Genome site. “We believe in the need for high-quality, curated information to educate physicians and give them confidence as they make treatment decisions for patients.”


Letter from the Editors

​The treatment of patients with cancer in the 21st century has evolved into a complicated algorithm, requiring knowledge of an individual patient’s tumor mutation status prior to initiating therapy. Making mastery of knowledge even more difficult, tumor mutational profiling studies have revealed a high degree of molecular heterogeneity among cancers, though they may appear similar at the histological level. Even within single genes, such as that encoded by EGFR in lung cancers, mutations can be associated with primary drug sensitivity, primary drug resistance, or acquired resistance to EGFR tyrosine kinase inhibitors, while other rarer EGFR mutations have less clear significance. Staying abreast of fast-paced research changes is difficult for time-pressed oncologists and medical caregivers. Knowledge about rare variants found in cancers is hard to track down, especially in busy clinics. As an increasing number of patients have their tumors genotyped, there will be—and is already—a dire need for an interactive, easily accessible educational tool that provides up-to-date information to physicians on the clinical relevance of mutations in cancers and mutation-specific clinical trial availability at the point of care. Until 2011, no such tool existed.

In January 2011, the Vanderbilt–Ingram Cancer Center (VICC) launched the nation’s first web-based precision cancer medicine knowledge resource, “My Cancer Genome,” to enable a genetically informed approach to cancer medicine. Created by Dr. Mia Levy and Dr. William Pao and containing content written by physicians and physician–scientists from around the world​, this online information tool is designed to quickly educate clinicians and others interested in the information. With just a few clicks, users of My Cancer Genome can get up-to-date information on the rapidly expanding list of genetic mutations that impact different cancers. Importantly, users can easily research various mutation-specific treatment and clinical trial options locally, nationally, and internationally.

The content is continually evolving. We started with cancers that are already recognized to have molecular heterogeneity and for which mutations already have relevance to existing and emerging targeted therapies. We plan to update the website regularly with new content, covering new genes and diseases. We welcome new contributors. No one person or institution can keep up with the pace and volume of data that is emerging. However, as a collaborative network, we can together move the field more quickly.

Because we believe that My Cancer Genome can eventually become self-sustaining, we have explored a wide variety of partnership opportunities. In February 2014, the Vanderbilt-Ingram Cancer Center and My Cancer Genome announced an agreement with GenomOncology, a technology company developing clinical tools to analyze genomic cancer data. GenomOncology holds the exclusive license to My Cancer Genome content, with non-commercial use of the My Cancer Genome website and mobile apps continuing to be open to the public. As part of the partnership, the groups are developing a decision support tool based on My Cancer Genome data. For more information, please see the APIs and Licensing page​.

Sincerely yours,

Mia Levy, MD, PhD, and Christine Lovly, MD, PhD​



Cancers covered



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Biotech Chinese and Israeli Strategic Collaboration: Pontifax and WuXi PharmaTech (Cayman) Inc. (NYSE: WX)

UPDATED on 12/15/2015

China’s WuXi raises a $290M VC fund with eyes on ‘cross-border’ biotech bets

By Damian Garde


WuXi PharmaTech, China’s largest CRO, closed an oversubscribed $290 million venture fund, turning its attention to biopharma startups at home and in the U.S.

Wuxi PharmaTech CEO Ge Li

The fund, which the company said exceeded its $200 million target, will bankroll investments in early-stage biotech and healthcare companies. WuXi’s first foray into VC, a $63 million fund debuted in 2011, bought the CRO stakes in 18 companies including U.S. biotechs Juno Therapeutics ($JUNO) and Agios Pharmaceuticals ($AGIO), plus Chinese upstarts Hua Medicine and Adagene.

Now WuXi wants to broaden its venture arm and deepen its presence in the growing biotech VC scene on two continents. The company plans to place its bets through deal-scouting offices in Shanghai and Boston, leaning on its fast-growing U.S. operation and decades of work in its native country.

“China and the United States are the two largest and most dynamic healthcare markets in the world and countries where our firm has deep investment expertise and experience,” WuXi Chief Financial Officer Edward Hu said in a statement. “The cross-border nature of our investment strategy and our appetite for early-stage innovation and entrepreneurship have aligned us well with the macro-trends in both countries.”

The move comes a week after WuXi abandoned its public listing and went private in a $3.3 billion deal led by founder and CEO Ge Li. The CRO, on pace for about $800 million in revenue this year, has been broadening its business model beyond traditional outsourced clinical trials, buying big into genomics and signing risk-sharing R&D deals with its pharma partners. And Li, joined by a syndicate of investors, believes its brightest future lies away from the public markets.

– read the statement

Related Articles:

WuXi Healthcare plots a $250M biotech venture fund for U.S., China

CRO giant WuXi is going private in a $3.3B deal

Biotech notches another $2B VC quarter, but can it last?


From: Gerard Loiseau <>

Date: Tuesday, December 15, 2015 at 12:38 PM

To: Aviva Lev-Ari <>

Subject: China !!

UPDATED on 11/5/2015

WuXi pads its revenue on the way to a big buyout decision

By Damian Garde

Wuxi PharmaTech CEO Ge Li

Chinese CRO WuXi PharmaTech ($WX) extended its run of quarterly growth on the eve of a shareholder vote that could take the company private in a multibillion-dollar deal.

In the third quarter, WuXi boosted its revenue 23.1% to $213.6 million, driven by 18% growth in lab services, a 19.6% jump in small-molecule manufacturing services and a 66% leap in biologics services. Profits, however, tumbled by nearly 50% to $16.1 million due largely to charges related to foreign exchange and losses tied to joint ventures with PRA Health Sciences ($PRAH) and AstraZeneca ($AZN), the company said.

WuXi is not providing a forward-looking guidance because it is preparing for the possibility of becoming a private company in the coming months. In April a group led by founder and CEO Ge Li made an offer to take the company off the market in a $3.3 billion deal. A special committee formed by WuXi’s board has voted in favor of the transaction, and the idea will come before a shareholder vote on Nov. 25.

If the deal is approved, WuXi will become part of a newly formed parent company through an all-cash transaction that trades $46 for each of WuXi’s American-traded securities. The total represents a 16.5% premium over WuXi’s closing price before the offer came to light.

Meanwhile, the company has continued to expand its business beyond traditional CRO work and more in line with Li’s long-stated vision of becoming “an open-access capability and technology platform that enables anyone and any company [to] discover and develop therapeutic products to benefit patients.” That has meant embracing genomics through its NextCODE subsidiary, which has signed deals with hospitals around the world to provide patient screening, and expanding its footprint to include capacity for cell therapies and other next-generation therapeutics.

WuXi AppTec Launches Representative Office in Israel, Forms Strategic Collaboration with Pontifax

SHANGHAI, Oct. 30, 2014 /PRNewswire/ — WuXi PharmaTech (Cayman) Inc. (NYSE: WX), a leading open-access R&D capability and technology platform company serving the pharmaceutical, biotechnology, and medical device industries, with operations in China and the United States, today announced the establishment of a representative office in the Tel Aviv area of Israel.

The new office will promote WuXi’s broad platform of integrated R&D services to local customers. It will also collaborate with Pontifax, a leading healthcare-dedicated venture capital firm based in Israel, to invest in promising technologies in Israel, particularly those that can potentially advance WuXi’s capabilities.

“We welcome WuXi’s presence in Israel and believe the new representative office will be mutually beneficial to WuXi and the Israeli biotech industry,” said Tomer Kariv, CEO of Pontifax.

“We are excited to establish a presence in Israel and to contribute to one of the most dynamic healthcare innovation ecosystems in the world,” said Dr. Ge Li, chairman and CEO of WuXi PharmaTech. “We value the expertise that Pontifax has developed in Israel’s biotech industry and look forward to working closely with them to help many of their portfolio companies and other startup companies. This step advances WuXi’s mission of helping entrepreneurs in the global life sciences industry to realize their dreams of developing innovative products to benefit the world’s patients.”

About WuXi PharmaTech

WuXi PharmaTech (NYSE: WX) is a leading open-access R&D capability and technology platform company serving the pharmaceutical, biotechnology, and medical device industries, with operations in China and the United States. As a research-driven and customer-focused company, WuXi PharmaTech provides pharmaceutical, biotechnology, and medical device companies with a broad and integrated portfolio of laboratory and manufacturing services throughout the drug and medical device R&D process. WuXi PharmaTech’s services are designed to help its global partners in shortening the cycle and lowering the cost of drug and medical device R&D. The operating subsidiaries of WuXi PharmaTech are known as WuXi AppTec. Please visit

For further information please contact:

Dana Yarden, MD, MBA
Executive Director, Israel Business Development
+972-9-9725617 or +972-54-8085692

Ronald Aldridge
Director of Investor Relations

Aaron Shi
Associate Director of Corporate Communications

SOURCE WuXi PharmaTech

WuXi PharmaTech

Web Site:


From: “PR Newswire for Journalists” <>
Sent: Thursday, October 30, 2014 5:42 PM

Corporate Profile

Services & Solutions by WuXi AppTec WuXi PharmaTech (pronounced woo-shee pharma-tek) is a leading global contract R&D services provider serving the pharmaceutical, biotech, and medical device industries. The company is headquartered in Shanghai and has operations in both China and the United States. We provide a broad and integrated portfolio of laboratory and manufacturing services throughout the R&D process. Our services are designed to help our global partners shorten the time and lower the cost of R&D. The parent company is known as WuXi PharmaTech, and its operating divisions are known as WuXi AppTec (pronounced woo-shee app-tek)
WuXi PharmaTech is the product of the merger in early 2008 of WuXi PharmaTech Inc., a chemistry-based company founded in China in 2000, and AppTec Laboratory Services Inc., a U.S. company founded in 2001 with expertise in medical-device and biologics testing. WuXi PharmaTech Inc. expanded its services rapidly throughout the decade, offering discovery chemistry services in 2001; process development in 2003; research manufacturing in 2004; bioanalytical chemistry in 2005; discovery biology in 2006; toxicology and formulation in 2007; commercial manufacturing in 2009; genomics, clinical trial management and research reagents in 2011; and biologics discovery, development and manufacturing in 2012.Biopharmaceutical and medical device research and development is complex, high-risk, and expensive for our customers. Improving R&D productivity is vitally important not only for the continued success of life sciences companies but also for the health of our families and each of us. Our competitive advantage rests on these elements:

  • an experienced international management team;
  • a highly educated and trained workforce of about 7,000 employees, including about 6,000 scientists, the majority with advanced degrees;
  • broad technical expertise;
  • operational excellence;
  • world-class facilities in both China and the United States;
  • an intense focus on a diversified, high-quality customer base;
  • a flexible contractual approach; and
  • strong procedures to protect customers’ intellectual property.

The company’s client list includes most of the major pharmaceutical and biotechnology companies. As our customers recognize the value we bring, they give WuXi AppTec larger and more valuable contracts. In recognition of the key contributions we made to their success, WuXi AppTec has received awards from leading pharmaceutical customers, including Pfizer, Merck, AstraZeneca, Novartis, Genentech, Millennium, and other companies.

WuXi is recognized as a strong growth company that has delivered solid financial performance since its inception. Revenues totaled $499.9 million and GAAP net income totaled $86.6 million in 2012. Our management has strategies in place to build on this record and to sustain long-term growth. Key drivers of growth in 2012 are our expanding capabilities and capacity and high-quality services in China-based Laboratory Services; increasing utilization of our integrated drug development services for API manufacturing, IND-enabling toxicology studies and IND filings with the China SFDA and global regulatory authorities; strong growth in testing revenues for both biologics and medical devices in our U.S.-based Laboratory Services; an expanding pipeline in both research manufacturing and commercial manufacturing; and the ramp-up of biologics drug discovery, development, and manufacturing services. Success in these areas is expected to deliver strong customer benefit and drive growth in shareholder value for many years to come. Our goal is to be the outsourcing partner of choice from bench to market.


Services and Solutions – WuXi AppTec – WuXi PharmaTech

Discovery Services

WuXi AppTec provides pharmaceutical discovery services across the entire spectrum of the drug discovery process. Our pharmaceutical discovery services can be fully integrated to provide a flexible and customized solution for client’s specific project needs.

Lab Testing Division (LTD)

Lab Testing Division (LTD) is comprised of seven business units. LTD’s integrated services and solutions in the fields of Chemistry and Biology span from early screening to preclinical development and into clinical sample analysis. Leveraging other established WuXi businesses in MedChem, synthesis and formulation, LTD is well positioned to enable customers to accelerate their discovery processes and empower them to bring new, innovative medicines to patients.

API Development and Manufacturing (STA)

Shanghai Syn-The-All Pharmaceutical Co. Ltd. (“STA”) is a wholly owned subsidiary of WuXi AppTec which provides an integrated platform with “end-to-end” small molecule APIs/intermediates development and manufacturing capabilities from preclinical to commercial stages. We proudly support over 100 life-science clients worldwide and manufacture over 100 APIs per year.

Development Services

WuXi AppTec provides end-to-end API services from process R&D, to API manufacturing at phase I, II, III and commercial scale. The services also include pre-formulation studies, analytical development, stability evaluation and formulation development, all the way to CMC services. All of these services are integrated to help our clients quickly and seamlessly move NCEs from preclinical stage to patients.

Biologics Services

WuXi AppTec provides a seamless, high-quality, single-source approach for the development, testing and manufacture of biotherapeutics. This single- source strategy can reduce the time-to-clinic and can significantly decrease the cost of our customers’ drug development efforts.

Medical Device Services

WuXi AppTec is uniquely positioned to support product development from concept to commercialization, with industry leading comprehensive testing programs that help ensure regulatory submission success.

Chemistry Services

WuXi AppTec offers a complete spectrum of chemistry services, all led by experts in their respective fields: from synthetic chemistry to chiral separations, from small molecule to peptide/peptidomimetics, from nucleoside to fluorinated building blocks, from milligram synthesis to kilogram GLP scale-up, and from reagent service to compound management.

Toxicology Services

WuXi AppTec’s toxicology services feature a full-range of in-vivo and in-vitro non-clinical safety evaluation programs. As the uniqueness of each product requires a case-by-case approach, we partner with clients to ensure that all study components meet specific program objectives.

Bioanalytical Services

WuXi AppTec offers comprehensive and FDA/OECD/SFDA GLP-compliant bioanalysis services to support preclinical and clinical development for small molecule drugs, biologics, vaccines and PD biomarkers.

Clinical and Regulatory Services

WuXi AppTec has strong experience in clinical trial management and regulatory affairs consultation; our experts are able to provide in-depth support to help clients bring new drugs and devices to the market smarter and faster.

Genome Center

WuXi Genome Center is a leading global genomic sequencing provider. It offers a complete solution to tackle biological and clinical challenges by combining components of genomics, bioinformatics, disease biology and clinical expertise to advance drug discovery, clinical development, and personalized medicine.

Biological Reagents

Abgent, a WuXi AppTec company, is a leading provider of antibodies and related services for biomedical research and drug discovery. Our competencies lie in the development of high quality antibodies and related reagents for the study of neurodegenerative diseases, stem cells, autophagy, and model organisms. Our antibodies are rigorously validated and optimized to ensure accurate and consistent performance.

For further information please contact:

Pontifax: Investor Details


18 Investments in 13 CompaniesExits

2 IPOs

– See more at:


Ran NussbaumTomer Kariv


Herzliya, Israel

  • Office

    8 Hama 3236 Nofim St.

    Herzliya Pituach

    Herzliya, 46725


– See more at:


Venture capital Firm

– See more at:

Current Team (3)


– See more at:

Founded: 2004
Type: Venture Capital that does Early Stage Venture, Later Stage Venture, and Private Equity InvestmentsSectors:Biotechnology, Health Care, Pharmaceuticals

Pontifax Ltd. is a venture capital firm specialzing in investments in incubation, seed or startups, early, and mid stage. It seeks to invest in life sciences sector. The firm seeks to invest in companies based in Israel

– See more at:

Categories favored by Pontifax

– See more at:

18 Investments in 13 CompaniesExits
Headquarters:HerzliyaDescription:Venture capital Firm

– See more at:

Arno TherapeuticsArno Therapeutics Private Equity

October 30, 2013
HeadSense MedicalHeadSense Medical Not Disclosed

July 8, 2013
Rewalk RoboticsRewalk Robotics Series D

June 17, 2013
TheraCoatTheraCoat Series A

May 21, 2013
cCAM BiotherapeuticscCAM Biotherapeutics Series A

September 15, 2012
Stimatix GIStimatix GI Not Disclosed

March 7, 2011
Avraham PharmaceuticalsAvraham Pharmaceuticals Series A

July 13, 2010
AposenseAposense Not Disclosed

May 23, 2010
Applied Immune TechnologiesApplied Immune Technologies Not Disclosed

April 26, 2010
ProtAbProtAb Series A

April 23, 2010
AposenseAposense Not Disclosed

August 20, 2008
CollplantCollplant Not Disclosed

April 1, 2008
CollplantCollplant Not Disclosed

April, 2007
CollplantCollplant Not Disclosed

February 13, 2007
CollplantCollplant Not Disclosed

September 4, 2006
CritiSenseCritiSense Not Disclosed

June 21, 2006
CritiSenseCritiSense Not Disclosed

June 8, 2005

– See more at:

– See more at:

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Introduction to Signaling

Curator: Larry H. Bernstein, MD, FCAP


We have laid down a basic structure and foundation for the remaining presentations.  It was essential to begin with the genome, which changed the course of teaching of biology and medicine in the 20th century, and introduced a central dogma of translation by transcription.  Nevertheless, there were significant inconsistencies and unanswered questions entering the twenty first century, accompanied by vast improvements in technical advances to clarify these issues. We have covered carbohydrate, protein, and lipid metabolism, which function in concert with the development of cellular structure, organ system development, and physiology.  To be sure, the progress in the study of the microscopic and particulate can’t be divorced from the observation of the whole.  We were left in the not so distant past with the impression of the Sufi story of the elephant and the three blind men, who one at a time held the tail, the trunk, and the ear, each proclaiming that it was the elephant.

I introduce here a story from the Brazilian biochemist, Jose

Eduardo des Salles Rosalino, on a formativr experience he had with the Nobelist, Luis Leloir.

Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it has changed from active form of the previous day to a non-active form. The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity. This assay was repeated with glycogen phosphorylase and the result was the opposite – it increases its activity. This led to the discovery

  • of cAMP activated protein kinase and
  • the assembly of a very complex system in the glycogen granule
  • that is not a simple carbohydrate polymer.

Instead, it has several proteins assembled and

  • preserves the capacity to receive from a single event (rise in cAMP)
  • two opposing signals with maximal efficiency,
  • stops glycogen synthesis,
  • as long as levels of glucose 6 phosphate are low
  • and increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat the assays with PK I, PKII and PKIII of M. Rouxii and using the Sutherland route to cAMP failed in this case. I then asked Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer) indicating this as his idea. The reason was my “chief”(SP) more than once, had said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things.” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.
Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved. This dialogue explains why during the reading and discussing “What is life” with him he asked me if as a biochemist in exile, talking to another biochemist, I expressed myself fully. I had considered that Schrödinger would not have confronted Darlington & Haldane because he was in U.K. in exile. This might explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes, in a way that suggested that the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of that year (1971).


Another aspect I think you must call attention to the following. Show in detail with different colors what carbons belongs to CoA, a huge molecule in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield

  • in comparison with anaerobic glycolysis.

The idea is

  • how much must have been spent in DNA sequences to build that molecule in order to use only two atoms of carbon.

Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it’s rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy).

Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

The discussions that follow are concerned with protein interactions and signaling.

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THE NEW ENGLAND VENTURE SUMMIT – Where Innovation Meets Capital  December 10, 2014  |  Hilton  |  Boston Dedham

Reporter: Aviva Lev-Ari, PhD, RN


FreeMind is pleased to announce its sponsorship and invite you to attend the New England Venture Summit presented by youngStartup Ventures


Come meet, interact and network with more than 500 VCs, Corporate VCs, angel investors, investment bankers and CEOs of early stage and emerging growth companies at the prestigious New England Venture Summit being held on December 10th, 2014 at the Hilton in Boston Dedham.


Whether you’re a startup seeking capital and exposure, or an investor seeking new deals, The New England Venture Summit presented by youngStartup Ventures – is one event you won’t want to miss.


A highly productive full-day venture conference, the New England Venture Summit is dedicated to showcasing VCs, Corporate VCs and angel investors committed to funding early stage and emerging companies.


This exclusive summit will feature a distinguished line up of more than 40 Investors on interactive panelspresentations by more than 50 companies seeking funding, and high-level networking opportunities.


Partial list of VCs confirmed to speak include:

John Albright, Co-Founder & Managing Partner, Relay Ventures | Grant Allen, Senior Vice President, ABB Technology Ventures | Jim Andelman, Managing Partner, Rincon Venture Partners | Gil Beyda, Managing Partner, Genacast Ventures | Eric Bielke, Senior Investment Associate, Siemens Venture Capital | Sim Blaustein, Principal, BDMI Fund | David Blumberg, Managing Partner, Blumberg Capital | Steven Chrust, Managing Director, Centripetal Capital Partners | Daniel Chui, Manager, Verizon Ventures | Andrew Clapp, Managing Partner, Arctaris Capital Partners | Charles Curran, Senior Director, Qualcomm Ventures | David Donabedian, Vice President, AbbVie Biotech Ventures | James Dugan, CEO & Managing Partner, OCA Ventures | Imran Eba, Partner, Action Potential Venture Capital | Rami Elkhatib, General Partner, Acero Capital | Kevin Ferro, CEO, Vatera Holdings | Alexander Galitsky, Managing Partner, Almaz Capital Partners | Rich Gliklich, XIR & Partner, General Catalyst Partners | Bosun Hau, Partner, MVM Life Science Partners | Laurence Hayward, Partner, Independence Equity | Ben Hemani, Analyst, Braemar Energy Ventures | Tetsuro Iwata, Senior Manager, MP Healthcare Venture Management | Shamez Kanji, General Partner, North Hill Ventures | Roman Kikta, Managing Partner, Mobility Ventures | Carey Lai, Director, Intel Capital | Al Lauritano, Director, Technology Licensing & Collaboration, BD Technologies | Jim Macdonald, Managing Director, First Analysis | Ignacio Martinez, Partner, Flagship Ventures | Vincent Miles, Partner, Abingworth | David Miller, Executive Managing Director, Clean Energy Venture Group | Jay Onda, Principal, DoCoMo Capital | Praveen Sahay, Managing Director, Wave Equity Partners | Akhil Saklecha, Managing Director, Artiman Ventures | Cory Steffek, Managing Director, Saudi Aramco Energy Ventures | Jason Tagler, Partner, Camden Partners | Sebastian Titz, Manager, New Ventures, 3M New Ventures | Tibor Toth, Managing Director of Investments, Massachusetts Clean Energy Center | Brent Traidman, General Partner, Fenox Venture Capital | Kathleen Utecht, Venture Partner, Core Innovation Capital | Geeta Vemuri, Senior Managing Director, Baxter Ventures | Sonali Vijayavargiya, Founder & Managing Director, Augment Ventures | David Ward, Managing Partner, MTI Ventures | Jan Westerhues, Investment Partner, Robert Bosch Venture Capital GmbH | Keith Witek, Corporate Vice President, Strategy and Corporate Development, AMD Ventures | Tim Wright, General Partner, Grandbanks Capital and many more.


Freemind has made special arrangement for our network to receive a special discount of 10% off the “early bird” rates.


To Register now and receive the special discount as well as take advantage of the “Early bird” discount rate, use the link below and enter discount code freemind by November 6th.


In addition to providing access to leading Investors, the conference will feature more than 50 pre-screened early stage companies seeking capital, and hardcore networking.


This conference will be attended by the best people in the industry. Please register early to avoid disappointment.



Get Noticed > Get Funded > Grow Faster


A select group of 50 Top Innovators will be chosen to present their breakthrough investment opportunities to an exclusive audience of Venture Capitalists, Private Investors, Investment Bankers, Corporate Investors, and Strategic Partners.


The deadline for presenting company applications is November 6th, 2014.


Apply to Present:

To be considered for one of the Top Innovator slots e-mail for an application.


Nominate a Startup:

To nominate your portfolio company or client send an email with contact details and any relevant information to


Questions about presenting? email us at





To RSVP by phone, to inquire about group rates or for more information call 212-202-1002


FEES: “Early bird” registration savings expire soon!  Use code “freemind” and receive an extra 10% off.

Entrepreneurs: Regular: $395 | At the Door: $790

Investors: Regular: $495 | At the Door: $990)

Service Providers: Regular: $695 | At the Door: $1,390)


Special Thanks to our Sponsors & Industry Partners:

Burns & Levinson LLP; Pepper Hamilton LLP; Dell | Intel; TriNet; Massachusetts Clean Energy Center; KPMG; Insperity; Abingworth; OCA Ventures; Arctaris Capital Partners; GetResponse; Center for Israeli Innovation; Israel Startup Network, SOS, ACTION; Indiana Health Industry Forum; FreeMind; Harvard Biotechnology Club & UVANY.


We hope you can join us for the exclusive forum.


FreeMInd  & youngStartup Ventures


To inquire about group rates, register by phone or for more information contact:

youngStartup Ventures at 212.202.1002

Check out highlight from previous summits here:

From: FreeMind Group <>
Reply-To: FreeMind Group <>
Date: Wed, 29 Oct 2014 18:00:14 +0000
To: Aviva <>
Subject: New England Venture Summit – Dec 10, Boston

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