Cutting Edge Researches In Applied

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Cutting Edge Researches In Applied Genomics of
Some Non Contagious Communicable Parasitic
Diseases
Cutting Edge Researches In Applied Genomics of
Some Non Contagious Communicable Parasitic
Diseases
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Life is a complex system for information storage
and processing. Information is transmitted from
cell to cell and from generation to generation.
Genetic information directs cellular function,
determines an organisms external appearance and
serves as the link between generations in every
species. A hereditary unit, later called a gene,
was found to determine a particular
characteristic or a trait of an organism.
A genome is all the DNA in an organism including
its genes. Genes carry information for making
proteins.These proteins determine among other
things, how the organism looks, how well its body
metabolizes food or fight infection and sometimes
even how it behaves.
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DNA, the genetic material is found principally
in the chromosome which is located in the nucleus
of a cell.
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It is formed by sequential joining of molecules
called nucleotides. Nucleotides in turn are
composed of three smaller molecules a phosphate,
a pentose sugar and a base.
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The nitrogenous bases fall into two types,
pyrimidines and purines.
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The sugar and phosphate components are constant,
what confers individually are the bases. Two
strands of the sugar phosphate components are
linked by pair of bases.
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Consequently DNA is double stranded that wound
round each other in a double helix.
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Each combination of three nucleotides code for a
particular amino acid.
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Called transcription and translation. The
transcription is the synthesis of three types of
RNA molecules messenger RNA, transfer RNA and
ribosomal RNA.
DNA
Transcription
mRNA
rRNA
tRNA
Ribosome
Translation
Cellular proteins
Ribosomal protein
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Of these mRNA can translate the genetic code
carried by DNA into proteins.
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However replication is the synthesis of DNA.
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Rarely a replication error occurs, the new
sequence of bases is not exactly like that of a
parental strand. Replication errors are a source
of mutation, a permanent change in a gene causing
a change in the phenotype.
The determination of the complete sequence of an
organism has captured the imagination of
researchers. Not only would we rapidly know the
minimal gene set necessary to make up an
organism, but we would also know the structure
and function of many genes. All diseases have a
genetic component, whether inherited or
resulting from the body response to
environmental stresses.
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Parasites remain some of the most important
human pathogens in the developing world. The
coevolution of parasite and its host, represents
all of the mechanisms they made to live with each
other. However such trait is considered a
reflection of differences in the genotype of the
hosts and parasites studied.
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There is a receptor-ligand interaction between
the parasite and hosts cell. Thus it appears
that many intracellular parasites utilize
distinct receptors on the surface of host cell
for recognition and entry. Blocking binding to
these receptors by antibodies may limit parasite
invasion and infection. In malaria this is a
major approach employed in vaccination against
established blood stage. Malaria merozoites
recognize a number of distinct receptors on the
erythrocyte surface including duffy blood group.
Antibodies against these receptors block cell
infection.
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The acquisition of immunity after infection with
eukaryotic parasites depends initially upon the
recognition of antigenic determinants or epitopes
by appropriate T lymphocyte receptor. This
involves prior handling and processing of antigen
by antigen presenting cell. The processed antigen
fragment binds to APC surface molecules codes by
genes in the MHC.
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The entire sequence of events is therefore under
tight genetic control, which is expressed through
two major groups of cell surface molecules, those
on the APC coded by MHC genes and those on the T
cell coded by the TCR genes. Some epitopes may
not be recognized either because the antigen
fragment doses not bind to the MHC molecules
(MHC) restriction) or because the epitope
revealed by binding to the MHC molecule dose not
bind to the TCR. The discovery of MHC restriction
of the immune response providing varying degrees
of protection against infectious pathogen. Also
parasitic resistance arises from antigenic
complexity of the parasites which present their
host with a wide array of antigenic molecules,
each of which can carry a number of distinct
epitopes. It is likely that several epitopes may
be functionally involved in initiating protective
response. Attempts are made to use defined
components of specific antigens as vaccine.
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Control of parasites is hampered by limitation
of current therapeutics, frequent reinfection, a
basic lack of understanding of many aspects of
parasite biology and a limited prospects for the
rapid introduction of effective vaccine. Partly
in response to this picture parasite genome
projects have been initiated. These projects
represent one of the most important developments
in our knowledge of parasites. However
translation of this knowledge into an
understanding of parasite biology and then on to
health care developments, needs efforts and
clarity of thoughts by scientists and funding
organizations.
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The road from gene identification to effective
treatment is long and fraught with challenges. In
the mean time, biotechnology companies are racing
ahead with commercialization by designing
diagnostic tests to detect errant genes in
parasites or hosts suspected of having particular
diseases or of being at risk of developing
them. Applied genomics has been established by
the application of cutting edge technology of
biological researches. This offers astounding
potential in molecular biology, immunology and
genetic engineering for the development of more
effective and longer lasting control tools of
target parasites and vectors.
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Rapid DNA sequencing have successfully
accelerated the identification of new genes. The
sequencing of random tags of genomic cDNA by
genomic sequence tage or expressed sequence tage
or the complete nucleotide sequence for important
organisms, are useful means for biological
understanding of the organisms.
Genome wide analysis of parasitic organisms is
at a very early stage, and it is unlikely that
the genome of parasites of economic importance
will be completely sequenced in the near future.
With limited resources it is not going to be
possible to determine the complete genome
sequence of all disease organisms, particularly
if they have large genomes.
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Because different organisms are evolutionarily
related and share some main biological mechanism,
the parasite genome projects have encouraged
comparative genomics based on the analysis of the
genome of closely or distantly related species.
The rationale behind this is that knowing the
gene function in one organism can help to infer a
possible function for the homologues gene in
other species. In this context, the knowledge of
genome of model organisms such as Caenorhabditis
elegans can be great relevance for understanding
gene function and gene regulation in related
organisms. The understanding of C.elegans genome
open new and exciting opportunities for
parasitology, since this model organisms is
closely related to parasites of economic
importance. Thus the genes which are targets for
new drugs and those responsible for genetic
resistance, identified in one species can be
sought in the second.
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The first stage towards validating candidate
gene for screen development is to understand the
function that they play in the life of the
parasite. While the second stage of target gene
validation includes the understanding of the
biochemical properties of the targeted genes.
This is very important in order to identify gene
targets with the potential to pick novel chemical
entities that posses high therapeutic index.
Importantly there are also several genes with no
functional identification that appear to be very
abundantly expressed. In these genes, we have a
message from the parasite, here are products
needed in abundance that might be useful drug
target or vaccine candidates.
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The WHO Training in Tropical Disease supported
international efforts to unravel the genome of
non contagious communicable disease especially
tropical parasites. Significant genome data were
accumulated, through successful collaborations
between scientists from developed and developing
countries and emphasis was put on rapid
availability of this information in public domain
databases. The genome projects took as its aims,
gene discovery for drug target and vaccine
candidate identification, genome mapping,
dissemination of genomic data to the world
community and training of endemic country
partners in genomic research.
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Schistosomiasis and Bancrofts filariasis are
two diseases, that lie at the top of Egyptian non
contagious communicable parasitic diseases. The
Brugia malayi and schistosome genome imitative
will place emphasis on using the wealth of
genome information and advances in functional
genomics to understand the mechanisms leading to
clinical disease and those essential for parasite
survival.
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Schistosomes have a comparatively large genome
estimated at 270 megabase pairs. To date only
S.mansoni has been analysed for genomic cloning
and chromosome localization. Currently there is
very little molecular information available for
S.haematobium. Extension of the EST programme to
this species will generate data for a major human
pathogen and provide valuable information about
the genetic diversity of schistosomes.
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Approximately 60 of the Schistosoma genome is
composed of repetitive sequences. Numerous
repetitive DNA elements have been identified
including satellite DNA, ribosomal RNA gene and
retrotransposon like sequences. The mitochondrial
DNA sequence in schistosomes has incorporated
the design of primers based to amplify the target
sequence. The schistosomes infectivity,
pathogenicity and immunogenicity are among the
biological features subject to genetic
variations. Actually, numerous stage and sex
specific genes, that elicit separate and
independent responses have already been
identified.
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Several epidemiological observations made on
residents of areas where S.mansoni are endemic
suggested the role of major gene in controlling
human susceptibility and resistance to this
parasite. First, predisposition to rapid and
severe reinfection had been observed in certain
individuals, after parasitological cure. Second,
epidemiological data showed that high infection
intensities were aggregated within certain
families rather than randomly distributed in the
population. Also the inflammatory granulomatous
response of the host to schistosomal antigens may
subside succeeded by tissue repair. However in
certain subject, it leads to extended and
irreversible fibrosis.
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Advanced fibrosis was more frequent in certain
families despite similar epidemiological
conditions for the whole study population. Also
fibrosis grades between parents and children
showed correlation, but those among spouses did
not. These observations have suggested that
inherited factors, controlled by distinct loci of
major genes might play a role in advanced
fibrosis in certain population. Segregation
analysis provided evidence for a codominant major
gene denoted as SM1 and SM2 that control the
level of infection by S.mansoni, taking into
account other risk factors for infection e.g.
water contact level, age and sex. Also evidences
for a direct association between the presence of
particular HLA alleles and hepatosplenomegaly
caused by S.mansoni in humans has been
accumulating steadily. This brings considerable
progress to the understanding of why only a
fraction of subjects infected by S.mansoni
develop severe fibrosis.
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To date the WHO Schistosome Genome Network is
one of the most wide spread international
collaborations. This network has been undertaking
collaborative studies on a) discovery and
characterization of new genes of S.mansoni and
S.japonicum, b) development of a physical map,
mainly for the S.mansoni genome, c) sequencing
and analysis of mitochondrial genome for both
species and development of new resources to be
used and distributed. The success of the SGP by
the EST strategy is demonstrated by the number of
catalogued genes that now reaches 15-20 of the
full gene complement of its genome. Conceivably
some of these may represent the expression of
private S.mansoni specific genes of importance
for the organism.
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Various attempts towards obtaining an effective
vaccine to eradicate schistosomiasis have been
done. In some cases, stage specific genes have
been used in immunological trials as potential
candidates. However no satisfactory effect had
been reached and the search for new stage
specific genes is still needed. The housekeeping
genes should be considered for further
investigations on their use for diagnosis,
chemotherapy and immunoprophylaxis.
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Lymphatic filariasis has recently been
recognized as the second leading cause of
permanent and long term disability world wide.
All filarial nematodes are closely related, and
so intensive study of one tractable species
should serve as a model for the others. B.malayi
was chosen in preference over the other medically
important filarial parasites for its ability to
be maintained by laboratory passage through jirds
and mosquitoes.
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The nuclear genome of B.malayi is about 100
million base pairs. This is about the same as
that of C.elegans. The repetitive DNA content at
approximately 15 is similar to that of
C.elegans, but the Brugia genome differ in that,
the nearly 10 of genome is made up of a single
tandemly repeated sequence, the HhaI repeat. This
repeat is found in all species of Brugia. This
repeat has served as the target sequence for a
hybridization, and a PCR based assay for the
sensitive detection of B.malayi parasites in
human blood. No such sequence have been
identified in W.bancrofti or C.elegans, although
many low copy numbers dispersed repeat sequences
including SspI repeat in W.bancrofti.
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In addition to the nuclear genome of Brugia
there are two other genomes within the parasite.
The first is the mitochondrial genome and the
second is the genome of the bacterial
endosymbiont. The endosymbiont might play roles
in nematode physiology and may be essential for
nematode survival and fecundity. This may lead
to the development of novel strategies to combat
the disease caused by filarial worms. Treatment
of jirds with tetracycline before infection with
B.malayi results in stunting of the worms and
failure to establish patent infections.
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Although the artful immunologist filarial worms
lived in milieu bathed in immune effectors cells,
yet can survive for many years. Each of a major
life cycle stages has unique immunological
characteristics.
Immune evasion genes of filarial parasites are
suite of molecules exposed on the surface of
adult worm and microfilariae. One important group
of them are the antioxidant product glutathione
peroxidase and superoxide dismutase. Also a
member of cystatin family that block a distinct
class of proteases. It can also block asparaginyl
endopeptidase which is important in intracellular
processing of antigens for presentation with MHC
class II.
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Also microfilariae secrete serine protease
inhibitor which is a potent inhibitor of two
neutrophil enzymes, cathepsin G and neurtophil
elastase. In addition B.malayi parasites encode
two sets of cytokines homologues of the immune
system genes. They are related to the
transforming growth factor ß that leads to the
down regulation of the host inflammatory
response, and macrophage migration inhibitory
factor, that may lead to suppression of
lymphocyte proliferation.
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To identify gene products critical to parasite
establishment in the host is to focus on the L3
or infective larva. A set of genes now designated
as abundant larval transcript or alt, may be
stockpiled by infective larvae for release in
mammalian host. The alt was tested in a jird
model of vaccination against B.malayi. It was
found that immunization with alt engendered 76
reduction in worm load. Another of the most
abundant transcript from B.malayi L3 is related
to a gene family first identified in Ancylostoma
caninum and secreted by its larvae when
stimulated to commence invasion of the host,
called Ancylostoma secreted protein antigen
(ASP). A near identical sequence of ASP has been
deposited as venom allergen homologue (VAL) in
B.malayi. A distant relative of them has been
found to inhibit neutrophil activation.
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The Filarial Genome Projects, will continue to
focus on new genes discovery, mapping development
of useful bioinformatic tools and training of
endemic country scientists. In addition the
complete sequence of Brugia mitochondrion and
wolbachia like endosymbiont are high priorities.
The ultimate goal of FGP is to provide resources
to the filarial research community. This will
support research culminating in the development
of new antifilarial drugs and/or vaccine against
lymphatic filariasis. The approach of sequencing
cDNA clones form many life cycle stages as
possible has proven to be extremely efficient in
identifying new gene.
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The EST database has proven exceedingly useful
in revealing, not only the obvious homologues of
important genes studied in other organisms but
also in discovering further new members of gene
families. The large scale EST effort should cover
many new genes and provide information about
genes involved in the biochemical pathways of the
nematode. Overall similarities between EST
profiles of various developmental stages may
contribute to a new understanding of the
interrelationships between different stages and
developmental pathways as well as host/parasite
interaction. The clustering has enabled us to
more accurately predict gene identification with
a novel regulatory elements and gene expression
that serve as valuable resource for chemotherapy
and vaccine design.
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To extend the discoveries made in Brugia to
other filarial pathogens, EST sequencing form
O.volvulus and W.bancrofti has been initiated.
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Now that many genome maps are available. Genetic
maps allow the genetic responses of parasite
population to be better characterised,
particularly where those responses may involve
selection at several loci scattered throughout
the genome. The ability to define the number of
loci responding to selection by anthelmintic and
the dominance relationship between alleles at
those loci is important basic information for the
mode of action of parasiticide compounds. A
strategy that aligns a well validated parasite
specific gene target with a highly diverse
collection of compounds promises to increase the
probability for the successful identification of
novel chemical entities with antiparasitic
properties.
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The announcements of promising scientific
achievements and more creative new
biotechnological approaches are necessary. Thus
it is imperative to encourage collaborations
between scientists working in public and academic
institutions where most of the information on
parasite genomics is produced. Also the
databases, that contain DNA and protein sequence
data, mapping data and other information of
genome analysis and biology of target parasites
are constructed. Together with network www sites
and e-mail mailing lists, would provide a forum
for communication within the wider
parasitological research communities.
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A resources of antiparasite drug target
definition and synthesis of biological
information relevant to vaccine effort may be
provided for carrying out milestone research
necessary for leading us to the welfare of life.
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