مجال
التميز
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إبداع علمي + تميز دراسي وبحثي
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جوائز التكريم:
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مسمى الجائزة:
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The Best Bio MSc
Student
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الجهة المانحة:
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Glasgow Caledonian
University
Graduation Ceremony
2013
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تاريخ
منح الجائزة:
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26/11/2013
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مجال التكريم:
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Best Bio MSc
Student
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البحوث المنشورة
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البحث (1):
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عنوان البحث:
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Functional analysis
of drug resistance-associated mutations in the Trypanosoma brucei adenosine
transporter 1 (TbAT1) and the proposal of a structural model for the protein
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رابط البحث:
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Click here
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تاريخ النشر:
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21/03/2015
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موجز عن البحث:
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The Trypanosoma
brucei aminopurine transporter P2/TbAT1 has long been implicated in the
transport of, and resistance to, the diamidine and melaminophenyl arsenical
classes of drugs that form the backbone of the pharmacopoeia against African
trypanosomiasis. Genetic alterations including deletions and single
nucleotide polymorphisms (SNPs) have been observed in numerous strains and
clinical isolates. Here, we systematically investigate each reported mutation
and assess their effects on transporter function after expression in a tbat1−/−
T. brucei line. Out of a set of six reported SNPs from a reported ‘resistance
allele’, none significantly impaired sensitivity to pentamidine, diminazene
or melarsoprol, relative to the TbAT1-WT allele, although several
combinations, and the deletion of the codon for residue F316, resulted in
highly significant impairment. These combinations of SNPs, and ΔF316, also
strongly impaired the uptake of [3H]-adenosine and [3H]-diminazene, identical
to the tbat1−/− control. The TbAT1 protein model predicted that residues F19,
D140 and F316 interact with the substrate of the transporter. Mutation of
D140 to alanine resulted in an inactive transporter, whereas the mutation
F19A produced a transporter with a slightly increased affinity for
[3H]-diminazene but reduced the uptake rate. The results presented here
validate earlier hypotheses of drug binding motifs for TbAT1
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البحث (2):
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عنوان البحث:
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9-(2′-Deoxy-2′-Fluoro-β-D-Arabinofuranosyl)
Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents
Transport-Related Drug Resistance
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رابط إلى البحث:
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http://aac.asm.org/content/61/6/e02719-16.abstract
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تاريخ النشر:
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3 April
2017
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موجز عن البحث:
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Current
chemotherapy against African sleeping sickness, a disease caused by the
protozoan parasite Trypanosoma brucei, is limited by toxicity, inefficacy,
and drug resistance. Nucleoside analogues have been successfully used to cure
T. brucei-infected mice, but they have the limitation of mainly being taken
up by the P2 nucleoside transporter, which, when mutated, is a common cause
of multidrug resistance in T. brucei. We report here that adenine arabinoside
(Ara-A) and the newly tested drug 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)
adenine (FANA-A) are instead taken up by the P1 nucleoside transporter, which
is not associated with drug resistance. Like Ara-A, FANA-A was found to be
resistant to cleavage by methylthioadenosine phosphorylase, an enzyme that
protects T. brucei against the antitrypanosomal effects of deoxyadenosine.
Another important factor behind the selectivity of nucleoside analogues is
how well they are phosphorylated within the cell. We found that the T. brucei
adenosine kinase had a higher catalytic efficiency with FANA-A than the
mammalian enzyme, and T. brucei cells treated with FANA-A accumulated high
levels of FANA-A triphosphate, which even surpassed the level of ATP and led
to cell cycle arrest, inhibition of DNA synthesis, and the accumulation of
DNA breaks. FANA-A inhibited nucleic acid biosynthesis and parasite
proliferation with 50% effective concentrations (EC50s) in the low nanomolar
range, whereas mammalian cell proliferation was inhibited in the micromolar
range. Both Ara-A and FANA-A, in combination with deoxycoformycin, cured T.
brucei-infected mice, but FANA-A did so at a dose 100 times lower than that
of Ara-A.
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البحث (3):
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عنوان البحث:
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Functional and genetic evidence that
nucleoside transport is highly conserved in Leishmania species: Implications
for pyrimidine-based chemotherapy
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رابط إلى البحث:
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http://www.sciencedirect.com/science/article/pii/S2211320716301208?via%3Dihub
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تاريخ النشر:
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20 April
2017
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موجز عن البحث:
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Leishmania pyrimidine salvage is replete with
opportunities for therapeutic intervention with enzyme inhibitors or
antimetabolites. Their uptake into cells depends upon specific transporters;
therefore it is essential to establish whether various Leishmania species possess
similar pyrimidine transporters capable of drug uptake. Here, we report a
comprehensive characterization of pyrimidine transport in L. major and L.
mexicana. In both species, two transporters for uridine/adenosine were
detected, one of which also transported uracil and the antimetabolites
5-fluoruracil (5-FU) and 5F,2′deoxyuridine (5F,2′dUrd), and was designated uridine-uracil transporter
1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2′dUrd and thymidine and was designated Nucleoside
Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like
genes encoding uridine/adenosine transporters, and an NT2 gene encoding an
inosine transporter, we cloned the corresponding L. major and L. mexicana
genes, expressing each in T. brucei. Consistent with the L. donovani reports,
the NT1-like genes of either species mediated the adenosine-sensitive uptake
of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes
mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine
antimetabolites tested, 5-FU and 5F,2′dUrd were the most effective antileishmanials;
resistance to both analogs was induced in L. major and L. mexicana. In each
case it was found that the resistant cells had lost the transport capacity
for the inducing drug. Metabolomics analysis found that the mechanism of
action of 5-FU and 5F-2′dUrd was similar in both Leishmania species, with
major changes in deoxynucleotide metabolism. We conclude that the pyrimidine
salvage system is highly conserved in Leishmania species – essential
information for the development of pyrimidine-based chemotherapy.
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البحث (4):
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عنوان البحث:
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Evaluation
of the antiprotozoan properties of 5′-norcarbocyclic pyrimidine nucleosides
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رابط إلى البحث:
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http://www.sciencedirect.com/science/article/pii/S0960894X17305334
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تاريخ النشر:
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17 May 2017
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موجز عن البحث:
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Carbocyclic nucleoside analogues have a
distinguished history as anti-infectious agents, including key antiviral
agents. Toxicity was initially a concern but this was reduced by the
introduction of 5′-nor variants. Here, we report the result of our
preliminary screening of a series of 5′-norcarbocyclic uridine analogues against protozoan
parasites, specifically the major pathogens Leishmania mexicana and
Trypanosoma brucei. The series displayed antiparasite activity in the low to
mid-micromolar range and establishes a preliminary structure-activity
relationship, with the 4′,N3-di-(3,5-dimethylbenzoyl)-substituted analogues
showing the most prominent activity. Utilizing an array of specially adapted
cell lines, it was established that this series of analogues likely act
through a common target. Moreover, the strong correlation between the
trypanocidal and anti-leishmanial activities indicates that this mechanism is
likely shared between the two species. EC50 values were unaffected by the
disabling of pyrimidine biosynthesis in T. brucei, showing that these uridine
analogues do not act directly on the enzymes of pyrimidine nucleotide
metabolism. The lack of cross-resistance with 5-fluorouracil, also
establishes that the carbocyclic analogues are not imported through the known
uracil transporters, thus offering forth new insights for this class of
nucleosides. The lack of cross-resistance with current trypanocides makes
this compound class interesting for further exploration.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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British Society for Parasitology Spring Meeting 2014
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تاريخ الإنعقاد:
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06
– 09 April 2014
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مكان
الإنعقاد:
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Cambridge, UK
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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Pyrimidine nucleoside transporters in L. major and
L. mexicana
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ملخص المشاركة:
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Research has
delineated four nucleobase and nucleoside transporters in Leishmania
promastigotes, designated as NT1-4 with the vast majority of studies on NT1
and NT2 being carried out using L. donovani genes. However, it is not known
whether the same genes mediate purine/pyrimidine transport in other
Leishmania species. In this study, we evaluated the uptake of 3H-uridine and
3H-thymidine in L. major and L. mexicana promastigotes. The uptake of both
nucleosides was mediated by LmexNT1, and LmajNT1, respectively. L. mexicana NT1 showed similarly efficient
transporter of uridine (Km =7.2±0.9 μM and Vmax =0.33±0.11 pmol∙107
cells-1∙s-1) and thymidine (Km =4.2±0.4 μM and Vmax =0.85±0.12 pmol∙107
cells-1∙s-1). Thymidine (Ki = 14.2±3.1) and adenosine (Ki = 0.23±0.04)
significantly inhibited uridine uptake. Transport of 3H-thymidine was also
inhibited by uridine and adenosine with Ki values of 6.0±0.6 μM and 0.25±0.04
μM, respectively. In L. major, LmajNT1 displayed high affinity for uridine,
with a Km value of 7.3±1.6 μM and Vmax of 0.078±0.005 pmol∙107 cells-1∙s-1,
which was inhibited by thymidine, 2’deoxyuridine and adenosine. 3H- thymidine
uptake showed a Km value of 30.7±2.1 and Vmax of 0.14±0.09, and was inhibited
by uridine and adenosine. These observations are all consistent with the L.
donovani model for nucleoside transport being conserved in L. major and L.
mexicana. We have now expressed L. mexicana and L. major NT1.1, NT1.2 and NT2
in a T. b. brucei strain lacking the aminopurine transporter TbAT1, in order
to confirm the genetic identities of the transport activities observed in
promastigotes.
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المؤتمر (2):
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عنوان المؤتمر:
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The 8th Saudi Students Conference
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تاريخ الإنعقاد:
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31st
January – 1st February 2015
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مكان
الإنعقاد:
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London, UK
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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Functional
expression of Leishmania nucleoside transporter genes
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ملخص المشاركة:
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Introduction
Purine and
pyrimidine nucleotides are absolutely necessary to all life which perform
many important functions in cells. Research has delineated four nucleobase
and nucleoside transporters in Leishmania promastigotes, designated as NT1-4
with the vast majority of studies on NT1 and NT2 being carried out using L.
donovani genes. However, it is not known whether the same genes mediate
purine/pyrimidine transport in other Leishmania species.
Aims
The main objective
was to characterize NT1.1, NT1.2 and NT2 in L. mexicana and L. major in order
to confirm the genetic identities of the transport activities observed in
promastigotes.
Methods: NT1.1,
NT1.2 and NT2 genes isolated from L. mexicana and L. major were expressed in
a T. b. brucei strain lacking the aminopurine transporter TbAT1 (T.b.b. B48).
Results
In contrast to B48
trypanosomes that were unable to transport inosine in the presence of
adenosine, introduction of LmaNT2 and lmexNT2 into B48 significantly
increased the rate of [3H]-inosine uptake to 0.00234 ± 0.0001pmol
(107cells)-1s-1 and 0.0023 ± 0.00032 pmol (107cells)-1 s-1, respectively. It
has also been proven that transport of uridine was mediated by LmexNT1A and
LmexNT1B in L. mexicana, and by LmaNT1A and LmaNT1B in L. major. While
uridine uptake was undetectable in T.b.brucei B48 in the presence of uracil,
introduction of NT1A and NT1B in T.b.brucei B48 significantly increased the
rate of uptake of [3H]-uridine to almost the same level as in L. major and L.
mexicana.
Conclusions
The results
confirmed that these genes are orthologues of the known and cloned L.
donovani ENT-family nucleoside transporters LdNT1 and LdNT2. These genes
provide an essential molecular tool for studying the role of transporters in
drug import and their potential involvement in the development of drug
resistance.
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المؤتمر (3):
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عنوان المؤتمر:
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British Society for
Parasitology Spring Meeting 2015
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تاريخ الإنعقاد:
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16 – 18 April 2015
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مكان
الإنعقاد:
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Liverpool, UK
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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Functional and
genetic evidence that nucleoside transport is highly conserved in Leishmania
species: implications for nucleoside-based chemotherapy
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ملخص المشاركة:
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In this study, we
characterized NT1A, NT1B, and NT2 genes in L. mexicana and L. major by
expressing them in a T. b. brucei strain lacking the aminopurine transporter
TbAT1. In contrast to B48 trypanosomes that were unable to transport
[3H]-inosine in the presence of adenosine, introduction of LmaNT2 and lmexNT2
into B48 significantly increased the rate of [3H]-inosine uptake to 0.00234 ±
0.0001pmol (107cells)-1s-1 and 0.0023 ± 0.00032 pmol (107cells)-1 s-1,
respectively. It has also been proven that transport of [3H]-uridine was
mediated by LmexNT1A and LmexNT1B in L. mexicana, and by LmaNT1A and LmaNT1B
in L. major. While [3H]-uridine uptake was undetectable in T. b. brucei B48
in the presence of uracil and inosine, introduction of NT1A and NT1B in T. b.
brucei B48 significantly increased the rate of uptake of [3H]-uridine to
almost the same level as in L. major and L. mexicana. The results confirmed
that these genes are orthologues of the known and cloned L. donovani
ENT-family nucleoside transporters LdNT1 and LdNT2. These genes provide an
essential molecular tool for studying the role of transporters in drug import
and their potential involvement in the development of drug resistance.
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المؤتمر (4):
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عنوان المؤتمر:
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The 9th Saudi Students Conference
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تاريخ الإنعقاد:
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13 – 14 Feb
2016
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مكان
الإنعقاد:
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Birmingham, UK
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طبيعة المشاركة:
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Poster Presentation
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عنوان المشاركة:
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Functional expression of Leishmania
nucleoside transporter
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ملخص المشاركة:
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Purine
uptake has been studied in detail in many cell types including protozoan
parasites. For the major protozoan pathogens most of the purine nucleoside
and nucleobase transporters – which also sometimes exhibit secondary
transport activity for pyrimidines – have been cloned. All of these
transporters belonged to the Equilibrative Nucleoside Transporter (ENT)
family. In stark contrast, comparatively little is known about the salvage of
preformed pyrimidines by protozoan parasites and the genes coding for
pyrimidine-specific transporters have not been identified. The principal aim
of this proposal is to identify the gene (family) encoding the protozoan pyrimidine
transporters using RNA interference target sequencing (RIT-seq) screens, and
next-generation RNA-sequencing (RNA-seq). Resistance to 5-fluorouracil (5-FU)
in T. b. brucei BSF s427- wild type and Leishmania mexicana promastigotes was
induced, and EC50 values were determined. Trypanosoma strains adapted to 5-FU
(Tbb-5FURes) displays 65-fold resistance compared to s427-wild type, and L.
mexicana adapted to 5-FU (Lmex-5FURes) was found to be 1145-fold resistant to
5FU compared to the wild type control. RNA-seq analysis was carried out to
analyse the transcriptome changes in both 5FU-resistant lines relative to
their sensitive controls, in order to identify genes potentially associated
with resistance to 5-FU in kinetoplastid cells. Sixteen genes were identified
as differentially expressed by RNA-seq, and the candidate genes were
overexpressed in resistant cells in order to confirm that the resistant
strains has restored the sensitivity to 5-FU. In addition, RIT-seq was
performed by exposing T. b. brucei expressing a genome-wide RNAi library to
5FU, which was used to select for RNAi fragments from the uracil transporter.
Therefore, what is now needed is to analyse the RIT-seq results carefully,
and possible candidate genes will be followed up by targeted RNAi knockdown
and other reverse genetics approaches. Efforts to identify the uracil
transporter genes are presently ongoing and identification of this gene will
significantly improve our understanding of the evolution of nutrient
transporters.
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المؤتمر (5):
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عنوان المؤتمر:
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British
Society for Parasitology
Trypanosomiasis and Leishmaniasis Seminar
2016
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تاريخ الإنعقاد:
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4-7
September 2016
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مكان
الإنعقاد:
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Czech republic
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طبيعة المشاركة:
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Poster Presentation
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عنوان المشاركة:
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Can folate/pteridine transporters transport
pyrimidine in protozoa
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ملخص المشاركة:
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Most
protozoa are capable of both salvaging performed pyrimidines and de novo
pyrimidine biosynthesis. This study seeks to identify the gene (family)
encoding the protozoan pyrimidine transporters using next-generation
RNA-sequencing (RNA-seq). Resistance to 5-fluorouracil (5-FU) was generated
in both T. b. brucei BSF s427- wild type and Leishmania mexicana
promastigotes, yielding clonal lines Tbb-5FURes and Lmex-5FURes,
respectively. RNA-seq was performed to identify and characterize the
differentially expressed genes between the sensitive and resistant strains.
Five folate/pteridine transporters genes (TbbPters) out of nine were
down-regulated in Tbb-5FURes, and two folate/pteridine genes (LmexPter) out
of eleven genes were down-regulated in Lmex-5FURes. Interestingly,
overexpression of TbbPter1 (Tb927.1.2820) in Tbb-5FURes caused a 3-fold
increase in 5-FU sensitivity (P< 0.0001). Overexpression of TbbPter1,
TbbPter3, TbbPter4 and TbbPter5 in Tbb-5FURes revealed a statistically
significant decrease (P<0.05) in sensitive to 6-azauracil, indicating a
restored uracil salvage pathway and decreasing reliance on de novo synthesis.
Characterization of LmexPters and generation of double gene deletions of
TbbPters (dKO) is ongoing, and transport and sensitivity assays will be
performed. Identification of pyrimidine transporter genes in protozoa will
aid in the therapeutic targeting of pyrimidine metabolism of protozoa.
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المؤتمر (6):
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عنوان المؤتمر:
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IV
Symposium of Tropical Health/COST Action
CM 1307 (WG3 and WG4) Joint Meeting
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تاريخ الإنعقاد:
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4-5 May
2017
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مكان
الإنعقاد:
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Pamplona, Spain
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طبيعة المشاركة:
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Oral Presentation
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عنوان المشاركة:
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Strategies to identify the genes encoding
pyrimidine-specific transporters in protozoa
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ملخص المشاركة:
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Most
protozoa are capable of both salvaging preformed pyrimidines and de novo
pyrimidine biosynthesis. This study seeks to identify the gene family
encoding the protozoan pyrimidine transporters using next-generation
RNA-sequencing (RNA-seq) and RNA interference target sequencing (RIT-seq).
The De Koning laboratory created 5-FU-resistant parasite lines of L. mexicana
(promastigotes) and of T. b. brucei (bloodstream forms) by in vitro exposure
to increasing concentrations of 5-FU. We carried out a comparative RNA-Seq
analysis of the parental wild-type strains and the 5-FU resistant lines of T.
b. brucei and L. mexicana in order to identify differences in gene
expression. We were particularly interested in any genes that encode for
proteins with at least 3 transmembrane domains that are significantly
down-regulated in the resistant lines Tbb-5FURes and Lmex-5FURes. In
addition, genome-wide RNAi library screens were performed in both pyrimidine
auxotrophic and prototrophic 2T1 cells exposed to 5-FU. High-throughput
RIT-seq implicated several strong hits from the 5-FU screens, which
apparently confer resistance to this pyrimidine analogue when their
expression is knocked down. We then compared the hits generated by RIT-seq
with the down-regulated genes in L. mexicana and T. brucei. Several candidate
pyrimidine transporters genes were identified. These results provide a valuable
resource for further exploration to identify the gene (family) encoding the
protozoan pyrimidine transporters that we know are expressed in Trypanosoma,
Leishmania, Trichomonas and other species. Functional expression, targeted
RNAi knockdown and reverse genetics of these candidate pyrimidine
transporters genes are in progress and will establish whether any of the
pyrimidine transport activities that we have previously identified in the
various protozoa is encoded by the genes under study. In conclusion, the
strategies proposed here are likely to lead to the identification of the
protozoan pyrimidine transporter genes. Identification of pyrimidine transporter
genes in protozoa will significantly improve our understanding of the
evolution of nutrient transporters.
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