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Research ArticleHuman Genetic Markers and StructuralPrediction of Plasmodium falciparumMultidrug ResistanceGene (pfmdr1) for Ligand Binding in Pregnant WomenAttending General Hospital Minna

B Lawal O K Shittu A Abubakar and A Y Kabiru

Department of Biochemistry Federal University of Technology PMB 65 Minna Nigeria

Correspondence should be addressed to B Lawal bashirlawal12gmailcom

Received 15 December 2017 Revised 17 March 2018 Accepted 1 April 2018 Published 16 May 2018

Academic Editor Francesco Pappalardo

Copyright copy 2018 B Lawal et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The study aims to determine the association of malaria infection with ABO blood groups and genotype and also to detect pointmutations at positions 86 184 1034 and 1042 of the Plasmodium falciparummultidrug resistance gene (pfmdr1) in blood samplescollected from pregnant women attending General Hospital Minna Out of 250 pregnant women screened 39 (1560) hadmalariainfection Prevalencewas higher inwomen during the third trimester (4615) genotypeAA (6410) andOblood group (5384)individuals when compared with others There was significant (119901 lt 005) decrease in Packed Cell Volume (PCV) hemoglobin(HGB) Red Blood Cells (RBC) and platelet (PLC) count in infected group when compared with noninfected group Althoughtwo of the isolates showed disrupted protein sequence at codon 1034ndash1042 no mutation was found in any of the P falciparumisolates Structural prediction of chemical ligand led to the identification of Neu5Ac1205722-3Gal1205731-31205731-4GlcGlcNAcThis compoundcan theoretically bind and change the functional integrity of the pfmdr1 protein thus providing a new window for malaria drugtarget

1 Introduction

Malaria is a vector borne infectious disease endemic inNigeria with about 97 of the population at risk [1] InNigeria malaria accounts for 60 of outpatient visits tohealth facilities 25 of childhood death and 11 of maternaldeath [2] The financial loss due to malaria annually isestimated to be about 132 billion naira in form of treatmentcost prevention and loss of man hours among others [3]Increased resistance of the Plasmodium falciparum parasiteto many of the available antimalarial drugs has made thetreatment of malaria increasingly difficult and poses a majorthreat to the malaria control and eradication program run-ning across the globe [4] The factors influencing the rate ofspread of antimalarial drug resistance include de novo muta-tion human population movement and infection amongmigrants drug use and malaria transmission intensity [5]More than any other issue poverty in adherence to drug

prescription and inadequate access to drugs continue to bemajor force in the development of resistance A wide rangeof unrecommended antimalarial medicines are available andsold over the counter in medicine stores and are accessibleto multigravid pregnant women in Nigeria [1] Healthcareproviders also prescribed unrecommended antimalarials dueto poor knowledge on best practices and low confidence inthe recommended drugs The impact of antimalarial drugresistance includes high mortality (resistant infections aremore often fatal) and morbidity Resistance to antimalarialdrugs has also increased the global cost of controlling thedisease and investigations on newer and more expensivedrugs Therapeutic failure necessitates consultation at ahealth facility for further diagnosis and treatment resultingin loss of working days for adults and absence from schoolfor children

Malaria during pregnancy is a major public healthconcern In most endemic areas pregnant women are the

HindawiJournal of Environmental and Public HealthVolume 2018 Article ID 3984316 13 pageshttpsdoiorg10115520183984316

2 Journal of Environmental and Public Health

main adult risk group for malaria [6] Pregnant women areparticularly susceptible to severe malaria and have a greaterrisk of antimalaria resistance due to loss of immunity duringpregnancy thus placing them at high risk of maternal andinfant morbidity and mortality abortion premature labourand stillbirth [7]

Genetic markers such as hemoglobin genotypes andblood groups have been associated with malaria but havebeen implicated with rosette formation and cytoadhesion [8]Variations in gene encoding functional glycotransferase havebeen associated with protection from P falciparum malariaAlso trisaccharide of ldquoArdquo and ldquoBrdquo blood group is presumedto act as receptor and function as an important factor forrosetting However the RBC of group O does not expresstrisaccharide blood types [9]

Genetic variations in pfmdr1 gene have played vital rolein malaria chemotherapy Mutations (N86Y Y184F S1034CN1042D and D1246Y) in the pfmdr1 gene have been asso-ciated with resistance to multiple antimalarial drugs such asquininemefloquine halofantrine artemisinin lumefantrineCQ and amodiaquine [10ndash13] However mutations in thisgene have been associated with geographic areas [14] andthere is a paucity of information on antimalarial drug resis-tance markers in Plasmodium falciparum infected pregnantwomen in Northern Nigeria Therefore it becomes relevantto determine the molecular marker of resistance in populaceof this region Also understanding the relationship betweenpfmdr1 amino acid sequence and its three-dimensional struc-tures will assist in predicting the chemical ligandsinhibitorthat can bind and change the functional integrity of theproteins This will provide information that is crucial indesigning an alternative and effective antimalarial drug thusproviding a new window for malaria drug target

2 Materials and Methods

21 Study Area This study was carried out in Minna NigerState located in the tropical zone of North Central NigeriaMinna the capital of Niger State Nigeria is located withinlongitude 6∘331015840E and latitude 9∘371015840N covering a land areaof 88 km2 with a population of 12 million (2006 PopulationCensus) Minna has a tropical climate with mean annualtemperature relative humidity and rainfall of 3020∘ 6100and 133400 cm respectively The climate presents two dis-tinct seasons a rainy season (AprilndashOctober) and dry season(NovemberndashMarch)

22 Inclusion andExclusionCriteria Allwomen enrolled hadearlier received intermittent preventive treatments (IPTp)with sulphadoxine-pyrimethamine (SP) as per current prac-tice The eligibility factor was set as women presentingwith gestational age 4 weeks and above to ensure adequatecoverage and data control Those who were not residentin the study area visitors and temporary residents whowere not officially registered for antenatal were not eligiblefor enrolment Other exclusion criteria included history ofadverse drug reaction or severe disease (such as hepatitisjaundice tuberculosis and obvious AIDS symptoms)

23 Ethical Considerations This study was approved by theEthical Committee of the Niger State Ministry of HealthMinna Nigeria Oral consent was obtained from the par-ticipant prior to inclusion in the study and before samplecollection All procedures were performed according to theguidelines for human experimentations in clinical researchas stated by the Federal Ministry of Health of Nigeria

24 Experimental Subjects and Sample Collection A total of250 women that attended antenatal care during the periodof study (December 2016) were recruited Each pregnantwoman was finger pricked using a sterile lancet to obtaincapillary blood for a rapid diagnostic test (RDT) for malariaSafety procedures were adopted in the collection of finger-prick blood samples by swabbing the area to be sampled with70 alcohol and allowing it to dry before collection Twomilliliters (2mL) of blood was then drawn (venipuncture)from malaria positive sample with a sterile disposable

25 Parasite Detection RDT based P falciparum specifichistidine-rich protein-2 (Bioline USA) was performedaccording to the manufacturerrsquos instructions to detect para-sites as recommended by the National Malaria Programme[15]

26 ABO Blood Grouping and Hemoglobin Genotyping TheABO blood group of each subject was determined using cellgrouping antisera (A B and D) as described by Simon-Okeet al [16] while the hemoglobin genotype separation wascarried out using cellulose acetate electrophoresis method asdescribed by Cheesbrough [17]

27 Hematological Parameters The hematological parame-ters hemoglobin (Hb) Packed Cell Volume (PCV) RedBlood Cells (RBC) White Blood Cells (WBC) and plateletscount (PLT) were determined using the automated hema-tologic analyzer SYSMEX KX21 a product of SYSMEXCorporation Japan as described by Dacie and Lewis [18]

28 Molecular Studies

281 DNA Extraction The genomic DNA was extractedfrom 2ml of infected blood using QIAmp DNA Mini Kit(Qiagen Hilden Germany) according to the manufacturerrsquosinstructions and subsequently stored at minus20∘C until use Thepresence of DNA was ascertained by subjecting it to 1agarose gel electrophoresis (DNA GelRed) and the purityof DNA was checked spectrophotometrically by calculatingthe A260A280 ratio

282 Amplification of pfmdr1 Gene Amplification of thepfmdr1 gene was performed on two fragments using poly-merase chain reaction (PCR) the first fragment containedcodons 86 and 184 the second fragment contained codons1034 and 1042 The primers were designed on the basis ofthe Plasmodium falciparum 3D7multidrug resistance protein(pfmdr1) partial mRNA sequence (GenBank accession noXM 0013517511) The reaction primers and conditions for

Journal of Environmental and Public Health 3

amplification of the first fragment (580 bp) were ForwardAGGTTGAAAAAGAGTTGAAC and Reverse ATGACA-CCACAAACATAAAT reaction conditions were 94∘C for3 minutes[94∘C for 30 seconds 45∘C for 1 minute 72∘Cfor 1 minute] times 30 cycles72∘C for 5 minutes The reactionprimers and conditions for amplification of the secondfragment (234 bp) were Forward GCATTT TATAATATG-CATACTG and Reverse GGATTTCATAAAGTCATCAACreaction conditions were 94∘C for 3 minutes[94∘C for 30seconds 55∘C for 1 minute65∘C for 40 seconds] times 30cycles65∘C for 5 minutes15∘C for 5 minutes [1 13]

The PCR amplification reactions were performed in a 20-120583l volume PCR tube containing 10U of TaqDNApolymerase(Pyro Hot Start) 250120583M each of the dNTPs 1x reactionbuffer with 15mM MgCl

2 1x stabilizer and tracking dye

and 4 120583l of genomic DNA All PCR reactions were carriedout in a thermal cycler (Techne TM Thermal cycler TC-312Fisher ScientificUK) PCRproductswere separated on a 15agarose gel (Invitrogen) and stained with ethidium bromidefor confirmation of amplification of the two pfmdr1 fragmentsas indicated by a band at 580 bp and 234 bp

283 Sequencing Protocol for pfmdr1 Polymorphisms inthe pfmdr1 gene were determined by direct sequencing ofthe amplicons resulting from the nested PCR using eachprimer for target gene amplification The sequencing wasdone using BigDye Terminator v31 cycle sequencing kit inan ABI 3730 sequencer (Applied Biosystems) The deducedamino acid sequences were aligned and analyzed with theLasergene5 software (DNASTAR Madison WI USA) usingthe reference sequences of 3D7 retrieved from Plasmodiumdatabase (httpwwwplasmodborg)

29 Bioinformatics Studies

291 Database Search The similarities in sequencedPFMDR gene were compared using pairwise alignmenttools (httpswwwncbinlmnihgov) and the nucleotidessequence was transcribed using sequence translationTranseq (EMBOSS) (httpswwwebiacuktoolsst) Thiswas compared with databased sequences using basic localalignment search tools (BLASTP) search default setting(httpswwwncbinlmnihgov) Five hits were selected formultiple sequences alignment using Clustal Omega methodBLOSUM matrix (httpwwwebacukmsatoolsclustalo)Specific features such as protein family domain cellularcomponent biological process and molecular function waspredicted using InterPro (httpwwwebiacukinterpro)

292 Receptor Protein Selection The PDBSum (httpwwwebacukpdbsum) and SAS databases were searched usingIPFMDR sequence to download the similar protein sequencefor structural prediction The best three hits were subjectedto multiple sequence alignment (httpwwwtcoffeecrgcat)(Laskowski 2001) The selected structure exists in chainsThe chains A were retrieved from httpwwwrcsborg forthe clustering and helical shapes alignment using ESPipt30 Advanced and all the hits proteins were model PyMOL(httpwwwpymolorg)

Table 1 Prevalence of malaria infection among different gestationperiods of pregnant women attending General Hospital Minna

Stages of pregnancy Frequency ()1st trimester 12 (3076)2nd trimester 9 (2307)3rd trimester 18 (4615)119905-value 491119901-value 0039

Table 2 Prevalence of malaria infection among different bloodgroups of pregnant women attending General Hospital Minna

ABO blood groups Frequency ()A 5 (1724)B 10 (2564)AB 3 (769)O 21 (5384)119905-value 242119901-value 0094

Table 3 Prevalence of malaria infection among different genotypeof pregnant women attending General Hospital Minna

Genotype Frequency ()AA 25 (6410)AS 14 (3589)SS 0 (00)AC 0 (00)119905-value 1609119901-value 0206

210 Statistical Analysis Data generated from this studywere analyzed using statistical package for social science(SPSS) version 16 and presented as means plusmn SE of the meanComparisons between different patient groups were carriedout by 119905-test The level of significance was set at 119901 lt 005

3 Results

31 ABO Blood Groups and Genotype Distributions Outof 250 pregnant women that were screened for malaria39 (1560) were found to be infected with Plasmodiumparasites as determined by RDT The prevalence of malariawas highest among third trimester (4615) as comparedwith the first (3076) and second trimester (2307) groups(Table 1) These differences were observed to be statisticallysignificant (119901 = 0039) All pregnant women that werepositive for malaria were also tested for ABO blood (Table 2)groups and genotype (Table 3) The prevalence was higheramong genotype AA (6410) than AS (3589) and thisdifference was not statistically significant (119901 = 0206) Thedistribution of ABO phenotypes among malaria infectionwas observed not to be statistically significant (119901 = 009)The highest proportion was among individuals with O bloodgroup (5384) followed by those with blood group B(2564) A (1724) and AB (769)


Table 4 Hematological parameters in Plasmodium falciparum infected pregnant women attending General Hospital Minna

Subjects PCV () HGB (gl) RBC WBC (times109L) Platelet (times103L)Malaria Positive(119873 = 20) 3356 plusmn 401 893 plusmn 109 597 plusmn 068 480 plusmn 032 26789 plusmn 2690

MalariaNegative(119873 = 10)

4450 plusmn 320 903 plusmn 147 701 plusmn 176 420 plusmn 045 59867 plusmn 3977

119901-value 00243 0195 0194 0271 0007Red blood cells (RBC) haemoglobin (HGB) packed cell volume (PCV) total white blood cell (WBC) and differential counts

Table 5 Protein sequence of Pfmdr1 gene isolates from genomic DNA of P falciparum infected pregnant patient sample

Sample code Primers used Protein sequence S1034C N1042D

A1 F AGGTTGAAAAAGAG AAC andR ATGACACCACAAACATAA

GTDYFCNLIEKAIDYKNKGQKRRI-IVNAALWGFSQSAQLFINSFAYWF-

GSXLIKRGTILVDDFMKXXW W


DYFCIlowastlowastKKLLIIKIKDKKEELLlowastMQL-YGDSVKALNYLLIVLPIGLDPSlowastLKE-

VLYlowastLMTLlowastNPDVFTlowastXD D

W wild type D disrupted protein sequence

Table 6 Amino acid and translated protein sequence of the pfmdr1 gene fragments

Amino acid sequence Translated protein sequence Blastp resultGGCACAGATTATTTCTGTAATTTGATAGAA-AAAGCTATTGATTATAAAAATAAAGGACAA-AAAAGAAGAATTATTGTAAATGCAGCTTTA-TGGGGATTCAGTCAAAGCGCTCAATTATTT-ATTAATAGTTTTGCCTATTGGTTTGGATCC-WTCTTAATTAAAAGAGGTACTATATTAGTT-GATGACTTTATGAAATSCA

GTDYFCNLIEKAIDYKNKGQKRRIIVNAALWGF-SQSAQLFINSFAYWFGSXLIKRGTILVDDFMKXX

BLASTP result wasused for multiple

sequence alignmentand further

bioinformatics datasearch

GATTATTTCTGTATTTGATAGAAAAAGCTATTG-ATTATAAAAATAAAGGACAAAAAAGAAGAA-TTATTGTAAATGCAGCTTTATGGGGATTCA-GTCAAAGCGCTCAATTATTTATTAATAGTTTTG-CCTATTGGTTTGGATCCTTCTTAATTAAAAGAG-GTACTATATTAGTTGATGACTTTATGAAATCCA-GATGTTTTTACATGAGA

DYFCIlowastlowastKKLLIIKIKDKKEELLlowastMQLYGDSVKALN-YLLIVLPIGLDPSlowastLKEVLYlowastLMT

LlowastNPDVFTlowastXNo data found

32Hematological Parameters The levels of PCVRBCHGBTWBC and PLC in malaria infected and noninfected preg-nant women attending General Hospital Minna are shownin Table 4 Test of significance (paired sample 119905-test) showeda significant differences in PCV and platelet count betweenmalaria positive and malaria negative pregnant women (119901 lt005) The 119901 value = 00243 and 0007 for PCV and plateletcount indicating a very significant decrease among malariainfected pregnant women

33 Prevalence of Drug Resistant Molecular Markers ThegenomicDNAof the 39 blood samples frommalarial infectedpregnant women were successfully isolated However outof the 39 genomic DNA samples only 16 samples frominfected women with resistance malaria were genotype forpfmdr1 gene by PCR and nested PCR out of which 12 (75)were successful while 4 (25) failed Thus pregnant womenwith antimalarial resistance had pfmdr1 gene prevalence of75 Amplification of the pfmdr1 gene was performed in

two different fragments Seven (7) samples were successfullyamplified in the first fragment (580 bp) containing codons 86and 184 six (6) samples were amplified from second fragment(234 bp) containing codons 1034 and 1042

34 pfmdr1 Gene Sequence Data Nomutation was observedat codon positions 86 184 1034 and 1042 of pfmdr1 in any ofthe P falciparum isolates However two of the isolates fromthe second fragment (sequence for 1034 and 1042) showeddisrupted protein sequence (Table 5)

35 Bioinformatics Database Search The amino acids andtranslated protein sequence of the of pfmdr1 gene isolatedfrom pregnant women attending General Hospital Minnaare shown in Table 6 Although pairwise alignment of thesetwo genes shows high (88) percentage similarities and 3gap between the sequences (Figure 5) BLASTP search of thesecond gene showed no significant similarity however the


CLUSTAL O(124) multiple sequence alignmentALS398191 DYFCNLIEKAIDYKNKGQKRRIIVNAALWGFSQSAQLFINSFAYWFGSFLIKRGTILVDD 60ETW199601 DYFCNLIEKAIDYKNKGQKRRIIVNAALWGFSQSAQLFINSFAYWFGSFLIKRGTILVDD 60ETW507511 DYFCNLIEKAIDYKNKGQKRRIIVNAALWGFSQSAQLFINSFAYWFGSFLIKRGTILVDD 60ETW534101 DYFCNLIEKAIDYKNKGQKRRIIVNAALWGFSQSAQLFINSFAYWFGSFLIKRGTILVDD 60ETW394681 DYFCNLIEKAIDYKNKGQKRRIIVNAALWGFSQSAQLFINSFAYWFGSFLIKRGTILVDD 60ETW317961 DYFCNLIEKAIDYKNKGQKRRIIVNAALWGFSQSAQLFIDSFAYWFGSFLIKRGTILVDD 60

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

ALS398191ETW199601ETW507511ETW534101ETW394681ETW317961

FMK 63FMK 63FMK 63FMK 63FMK 63FMK 63lowastlowastlowast

Figure 1 Similarities of the isolated pfmdr1 gene with those reported from other African countries

first gene produced multiple hits out of which five (5) hitswere selected for multiple sequence alignment The resultsshow very high similarities of the gene with those reportedfrom other African countries (Figure 1)

36 Receptor Protein Selection The PDBSum search ofIPFMDR sequence retrieves seven (7) hits of similar proteinsequence for structural prediction (Table 7) However 4RHSSPLTB STT and 5LUQ are the most similar protein with 317and 348 percentages

T-Coffee and ESPript 30 show multiple sequences align-ment of pfmdr1 homologous protein of crystal structureof an inward-facing eukaryotic ABC multidrug transporter(IEMDT) crystal structure of pltb (SPLTB) and structureof typhoid toxin (STT) The T-Coffee alignment showspercentage similarity of homologous protein IEMDT SPLTBand STT to be 37 64 and 65 respectively (Figure 2) whileESPript 30 shows the 3-dimensional helical folding of thehomologous protein (Figure 3) IEMDT has twenty-one 120572-helices twelve 120573 strands and two n while SPLTB and STThave two 120572-helices and five 120573 strands (Figure 4)

4 Discussion

Pregnant women living in malaria endemic regions par-ticularly in sub-Saharan Africa are associated with a highfrequency and density of Plasmodium falciparum parasitewith high rates of maternal morbidity [19] The prevalenceof malaria demonstrated in this study was 1560 lower than52 and 882 reported among pregnant women attendingantenatal clinic in southwest Nigeria [20 21]

Previously the reports of the prevalence of malaria inpregnancy were variable and high inNiger State where preva-lence rates between 582 and 8340 were documented [2223] High prevalence of malaria in pregnancy (41) has beenattributed to poor compliance to the use of insecticide treatednets and intermittent preventive therapy [24] However thesereports contrast sharply with the findings of this study wherea prevalence rate of 1560 was obtained for the pregnantwomen attending General Hospital Minna This finding is

not surprising as all women enrolled in this study hadearlier received IPTp with SP as per current practice inNiger State Previous researchers have also shown strongcorrelation between the use of long lasting insecticide treatednets (LLIN) and reduction in prevalence of parasitaemia andanaemia in pregnant women stillbirths and improvementsin birth weights of babies [25 26] Thus the low preva-lence rate recorded in this study may be attributed to theimproved understanding and compliances of the antenatalclinic women about malaria control strategies like use ofLLIN andor alternative intermittent preventive treatmentwith pyrimethamine sulfadoxine (SP) In addition the lowerprevalence obtained in this study might be due to the factthat this study was carried out during the dried seasonas opposed to the works of Omalu et al [22] and Ejimaet al [23] that were carried out during raining seasonThis finding agrees with Ayanda [27] who opined thatprevalence of P falciparum infection is higher in the wetseason than in the dry season while Minakaw et al [28]reported that rainy season presents favorable environmentalconditions that enhance mosquito breeding and survivalthrough the proliferation of larval habitats and improvedhumidity respectively

Various researchers have reported high seroprevalence ofmalaria at different trimesters of pregnancy [29] Howeveraccording to gestational age of pregnancy this study recordedthe highest seroprevalence rate in the third trimester followedby the first trimester and least was recorded among subjectsin their second trimester This finding agrees with the worksof Idowu et al [29] Ejima et al [23] and Omalu et al[22] which recorded high seroprevalence in third trimesterand least in second trimester but does not correlate withthe work of Brabin [30] who reported higher prevalencein the second trimester of pregnancy while Allesandro andLangerock [31] and Obianumba [32] identified higher riskof malaria in the first trimester of pregnancy Howeverthe 3rd trimester being of highest seroprevalence rate ofmalaria infection as obtained in this study suggests that thepregnantwomenhave significant loss of immunity during latepregnancy


Table7PD

BSum

database

inform

ationof

pfmdr1g

eneh

omologou

sprotein

sequ

enceSequenceGTD

YFCN

LIEK

AID

YKNKG

QKR

RIIV

NAALW

GFS

QSA

QLF

INSFAY

WFG

SXLIKR

GTIL-

VDDFM

KXX

Sequ

ence

leng

th67resid

ues

PDB

code

Mod

elLeng

th-ta

geidentity

aa

overlap

119911-score

Ligand

sProteinname

13w

mg(A)

X-ray240

A589

273

441398

DMU

TRS

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter

g277va278va279vmutant

incomplex

with

acyclic

peptideinh

ibito

raC

AP

23w

mf(A

)X-

ray260

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


3IEMDT(A)

X-ray275

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter

44rhs(A

)X-

ray19

2A114

317

411269

ACTSIA-

SIA-

GAL

Crystalstructure

ofGD2

boun

dpltb

5SP

LTB(A)

X-ray208

A114

317

411269

ACT

Crystalstructure

ofpltb

6ST

T(A)

X-ray239

A114

317

411269

GOL

Structureo

ftypho

idtoxin

75luq

(A)

X-ray430

A3725

348

461116

Crystalstructure

ofhu

man

DNA-

depend

entp

rotein

kinase

catalytic

subu

nit

(DNA-

PKcs)


BAD AVG GOOD

PFMDR1 69SPLTB 68STT 68IEMDT 38cons 69

PFMDR1 ------------------------------------------------------------------------SPLTB -----------EWTGDNTNAY---------------------------------------------------STT -----------EWTGDNTNAY---------------------------------------------------IEMDT TGVTARRIFALAWSSSATMIVIGFIASILEGATLPAFAIVFGRMFQVFTKSKSQIEGETWKYSVGFVGIGVF

cons

PFMDR1 ----------------------------------------------------------GTDYFCNLIEKAIDSPLTB --------------YSDEVISELHVGQIDTSPYFCIKTVKANGSGTPVVACAVSKQSIWAPSFKELLDQARYSTT --------------YSDEVISELHVGQIDTSPYFCIKTVKANGSGTPVVACAVSKQSIWAPSFKELLDQARY

EFIVAGSRTALFGIASERLARDL-----------------------------------RVAAFSNLVEQDVT

cons

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------

YFDRRKAGELGGKLNNDVQVIQYSFSKLGAVLFNLAQCVVGIIVAFIFAPALTGVLIALSPLVVLAGAAQMI

cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------

EMSGNTKRSSEAYASAGSVAAEVFSNIRTTKAFEAERYETQRYGSKLDPLYRLGRRRYISDGLFFGLSMLVI

cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------

STT ------------------------------------------------------------------------FCVYALALWWGGQLIARGSLNLGNLLTAFFSAILGFMGVGQAAQVWPDVTRGLGAGGELFAMIDRVPQYRRP

cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------

DPGAEVVTQPLVLKQGIVFENVHFRYPTRMNVEVLRGISLTIPNGKTVAIVGGSGAGKSTIIQLLMRFYDIE

cons

lowast

lowast

lowast lowast

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------

PQGGGLLLFDGTPAWNYDFHALRSQIGLVSQEPVLFSGTIRDNILYGKRDATDEEVIQALREANAYSFVMAL

cons

PFMDR1 -------------YKNKGQKRRIIVNAALWGFSQSAQLFINS-FAYW-------------------------SPLTB -------------FYSTGQSVRIHVQKNIWTYP----LFVNT-FSA--------------------------STT -------------FYSTGQSVRIHVQKNIWTYP----LFVNT-FSA--------------------------

PDGLDTEVGERGLALSGGQKQRIAIARAILKHP----TLLCLDESTSALDAESEALVQEALDRMMASDGVTS

cons

PFMDR1 -------------------FGSXLIKRGTILVDDFMKX------------XSPLTB ---------------------NALVGLSSCSATQCFGP------------KSTT ---------------------NALVGLSSCSATQCFGP------------KIEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

VVIAHRLSTVARADLILVMQDGVVVEQGNHSELMALGPSGFYYQLVEKQLA

cons

lowastlowast lowastlowast

Figure 2 T-Coffee multiple sequences alignment of pfmdr1 homologous protein


W TSASGPESAYTTGVTARRIFALA SSA MIVIGFIASILEGATLPAFAIVFGRMFQVFTKSW TTE GDN NW TTE GDN N

TT TT

KSQIEGETWKYSVGFVGIGVFEFIVAGSRTALFGIASERLARDLRVAAFSNLVEQDVTYF

DRRKAGELGGKLNNDVQVIQYSFSKLGAVLFNLAQCVVGIIVAFIFAPALTGVLIALSPL

AY S IV NVVLAGAAQMIEMSGNTKRSSE A AGS AAEVFS RTTKAFEAERYETQRYGSKLDPLAY S II Q Y DEV SELHVG AY S II Q Y DEV SELHVG

FCV LYRLGRRRYISDGLFFGLSMLVI YA ALWWGGQLIARGSLNLGNLLTAFFSAILGFMGFCI VDTSPY KT KFCI VDTSPY KT K

TT

TT

VGQAAQVWPDVTRGLGAGGELFAMIDRVPQYRRPDPGAEVVTQPLVLKQGIVFENVHFRY

TT TT TT TTT TT

G GS II L T APTRMNVEVLRGISLTIPNGKTVAIVGG A KST QLLMRFYDIEPQGGGLL FDG PG GN VA A Q IA S TPV C VSK SG GN VA A Q IA S TPV C VSK S

TT TT

TT

W F LHNYD A RSQIGLVSQEPVLFSGTIRDNILYGKRDATDEEVIQALREANAYSFVMALPDW F LKAPS E LDW F LKAPS E LD

TT

S GQ RI I P SI R L T AL A VQGLDTEVGERGLAL G KQ A A A LKH TL CLDES S D E EAL EALDRMMAS GQ RI I P SV K V N LV L ATQARYFY T SV H Q N WTY LF NTFSA A G SCS QCFS GQ RI I P SV K V N LV L ATQARYFY T SV H Q N WTY LF NTFSA A G SCS QCF

TT TT

GSDGVTSVVIAHRLSTVARADLILVMQD VVVEQGNHSELMALGPSGFYYQLVEKQLASGDG PKG PKLEHHHHHH

MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600

IEMDTSTTSPLTBSPLTBIEMDT

6 1 0IEMDTSTTSPLTBSPLTB

1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2

Figure 3 Clustering and helical shapes alignment of pfmdr1 gene homologous protein Included proteins are crystal structure of an inward-facing eukaryotic ABC multidrug transporter crystal structure of pltb and structure of typhoid toxin The secondary structure elements areas follows 120572-helices are shown as large coils 310 helices are shown in small coils labeled 120578 120573 strands are shown in arrows labeled 120573 and 120573 turnsare labeled TT The identical residues are shown on a red background with conserved residues in red and conserved regions in blue boxes


(a) (b)

(c)

Figure 4 PyMOL build structure of (a) STT monomers (b) SPLTTB monomer and (c) SPLTTB pentamer

NW Score Identities Gaps Strand Frame

311 190216(88) 7216(3) PlusPlusFeatures

Query 1 GGCACAGATTATTTCTGTAATTTGATAGAAAAAGCTATTGATTATAAAAATAAAGGACAA 60

Sbjct 1 G------ATTATTTCTGTA-TTTGATAGAAAAAGCTATTGATTATAAAAATAAAGGACAA 53

Query 61 AAAAGAAGAATTATTGTAAATGCAGCTTTATGGGGATTCAGTCAAAGCGCTCAATTATTT 120

Sbjct 54 AAAAGAAGAATTATTGTAAATGCAGCTTTATGGGGATTCAGTCAAAGCGCTCAATTATTT 113

Query 121 ATTAATAGTTTTGCCTATTGGTTTGGATCCWTCTTAATTAAAAGAGGTACTATATTAGTT 180

Sbjct 114 ATTAATAGTTTTGCCTATTGGTTTGGATCCTTCTTAATTAAAAGAGGTACTATATTAGTT 173

Query 181 GATGACTTTATGAAATSCA 199

Sbjct 174 GATGACTTTATGAAATCCAGATGTTTTTACATGAGA 209

Figure 5 Pairwise alignment of the isolated pfmdr1 gene sequences


The notion of pregnancy as an altered state of immunesuppression is well documented [33 34] Third trimester ofpregnancy is a time period that poses a risk of increasedsusceptibility to parasitic and infectious diseases since thematernal immune system is solely responsible for defendingagainst infections and protecting the fetus because both thefetal and the placental responses are limited [35] This mayhave been the reason for the higher susceptibility to malariaby women in their third trimester of pregnancy as recordedin this study

Therefore the pregnant women in this area may have evi-dence of malarial infection at the time of birth if proper con-trol and preventive measures are not taken thus increasingthe susceptibility of their offspring to incidence of congenitalmalaria low birth weight and mortality In support of thisclaim Omalu et al [36] reported 263 congenital malaria461 placental malaria and 592 cord malaria with lowbirth weight but no mortality in this region (Minna NigerState) Similarly 510 prevalence of congenital malaria wasrecorded at Ibadan University College Hospital [37] 4670at Ile-Ife Southwestern Nigeria [38] and 1300 at CalabarTeaching Hospital [39] Also the observation with secondtrimester could be a result of constant intermittent preventivetreatments in pregnancy (IPTp) given to pregnant womenduring antenatal care visit which usually commence duringsecond trimester [32]

Rapid diagnostic test (RDT) has been recommendedto improve diagnostic efficiency which is important forpreventing indiscriminate use of ACT thereby preventing ordelaying the development of parasite resistance to this newfirst-line drug [40] Also RDTs can be used as a stop-gapwhen microscopy services are not operating or as a primarydiagnostic tool for ruralremote areas without microscopyservices [41] The sensitivity of RDT reported in this study(75) is lower than 100 sensitivity previously reported inNigeria [42] and 96 97 and 976 reported in ZambiaZanzibar and Thailand respectively [40 43 44] Howeverpercentage of patients that were false positive (25) recordedin this study is considerably high This suggests that therewere chances of RDT to classify a healthy patient as beingsick Ayogu [45] reported a very low (32) failure of PCRconfirmation of malarial patient who tested positive by RDTThe patients with false positive result by RDT are likely tobe patients with persistently circulating antigen due to prioruse of antimalarial However other factors not investigatedin this work such as age differences in sensitivity to RDTrheumatoid factor cross-reacting in the blood [46] and cross-reactivity with heterophile antibodies [47] can be responsiblefor false positive result in RDT It is therefore necessaryto consider clinical situations and laboratory and especiallymicroscopic confirmation tests in cases of suspected falsepositive results for malaria RDT [48]

Prevalence of malaria parasites seemed to be relativelyhigh across a blood group O (5384) and AB (769)subjects recording the least infection rates High prevalenceof infection in blood group O shows that they are most sus-ceptible to uncomplicated malaria infection This disagreeswith the findings of Simon-Oke et al [16] who reported thatprevalence ofmalaria parasites was highest among those with

AB blood group (600) and lowest in those with B bloodgroup (372)

Natural selection for resistance against malaria may favorblood group O as it protects against severe P falciparummalaria [49] However it should be clear that the high occur-rence of malaria in blood group O in this study populationis not surprising as the finding does not disapprove thehypothesis about a selective survival evolutionary advantageof P falciparum infection in blood group O compared withnon-O blood groups in malaria endemic areas but ratherfurther explains the hypothesis that O blood type groupsare more susceptible to uncomplicated malaria but lesssusceptible to complicated malaria This corresponds to theresult of Rowe et al [8] in Mali where blood group O had ahigher prevalence of uncomplicated malaria

There are numerous publications on the strong protec-tion of the HbAS trait against mild and severe falciparummalaria However this study shows that pregnant womenwith genotype ldquoAArdquo were most susceptible to malaria with6410 of the total infection compared to AS (3589)Theseare not surprising as similar occurrences have been reportedin other parts of Nigeria and other African countries [50ndash56] It has been recorded by Friedman [57] that the loweringof oxygen which causes sickling shape in the blood of SSand AS people reduces the parasite growth and can causethe parasites to die but this is not so with the AA genotypeGenotype AA is the most prevalent genotype in this part ofthe world and more prone to malaria infection than othergenotypes because of the absence of any sickle cell moleculesin the blood

Anaemia in falciparum malaria is mainly due to thedestruction of parasitized red cells However according toOgbodo et al [58] increased parasite density ultimatelyleads to increase in red cell breakdown and consequentlyanaemia Brabin [30] reported 70ndash80 of pregnant womenin malaria area are susceptible to anaemia However basedon the definition of anaemia in pregnancy which is PCV ofless than 30 [59] infected women in this study cannot beclassified as being anaemic (as the mean PCV was gt30)These findings corroborate with those of similar studieswhich reported PCV level of 3016plusmn555 [60] and 3356plusmn048 [61] in pregnant women However the mean PCVRBC and HGB among malaria infected mothers were lowerthan mean PCV RBC and HGB of those without malariaparasite which can be suggestive of vulnerability to anaemiain pregnancy thus placing them at higher risk of morbiditiessuch as congestive heart failure fetal demise and mortalityassociated with hemorrhage at the time of delivery [62]

The use of advanced molecular techniques is extremelyuseful for the detection of drug resistance in malarial para-sites and plays an immense role in the epidemiological surveyas well as in regular updating of the antimalarial drug policyregimes [4] Therefore it plays an important role in hospital-based prevalence study tomonitor the drug resistance Its usein detection of the point mutation in the pfmdr1 gene in thisstudy reports no mutation detection at codon positions 86184 1034 and 1042 of pfmdr1 in P falciparum isolates frompregnant women in Minna Niger State Northern Nigeriasuggesting that these alleles are not a marker for multidrug


resistant Plasmodium falciparum in Niger State Nigeria butlarger sample size should conducted

Plasmodium falciparum multidrug resistance gene 1(pfmdr1) is an adenosine triphosphate-binding cassette pro-tein located on the parasitersquos food vacuole and thus facilitatestransport of solutes into the cell In sensitivity to antimalarialdrugs pfmdr1 acts as an active drug transporter thus quinineand possibly other antimalarial drugs occupy the commondrug binding site of pfmdr1 thereby inhibiting transport ofother solutes Mutations in this gene are known to alter thebinding of this drug thereby allowing the flow of solute viaparasitersquos food vacuole and thus drug resistance developed[63] Therefore the prediction of the structure pfmdr1 geneis important for identification of an analogous compoundwhich can appropriately fit into the parasite food vacuole andblock the flowmovement of solute into the parasite therebysuggesting new ligand for drug target modification

Basic local alignment search tool (BLAST) is the toolmostfrequently used for calculating sequence similarity [64] Thetable of BLAST hits is a section showing all of the alignmentblocks for each BLAST hit The sequence alignments showus how well the pfmdr1 gene isolated matches pfmdr1 genereported in the database for other African countries It hasbeen reported that BLAST results with sequence identityranges of 75ndash92 have high degree of sequence homology[65] In this study it was noted that the five hits match muchbetter the query pfmdr1 sequence than the remaining BLASThits and the MSA of these genes shows very high similaritiesof the gene with those reported from other African countriestherefore suggesting that a particular ligand could be pre-dicted to target pfmdr1 gene in P falciparum there by openinga novel window for malaria drug discovery

It has been reported that molecular interaction andfunction of a protein depend on its three-dimensional struc-ture [64] Thus prediction of protein structures assists inpredicting the chemical ligandsinhibitor that can bind andchange the expression or inhibit its expression thus providinga new window for drug target

On the basis of this pfmdr1 gene was searched forsimilar protein sequence (homologous protein sequence) andstructure prediction through PDBSum databases However4RHS SPLTB STT and 5LUQ are the most similar proteinswith 317 and 348 percentages T-Coffee and ESPript 30 showthat there is a good alignment and orientation of pfmdr1 genesequence with crystal structure of pltb (SPLTB) and structureof typhoid toxin (STT) This suggested that this gene sharesstructural identity

T-Coffee has been reported to be definitely superior toother MSA programs with regard to alignment accuracy inall datasets [66] Its alignment similarities ranges of 58ndash91have been considered to be of high degree [67] In this studyalignment of pfmdr1 shows a high degree of homology to theSTT (65) and SPLTB (64) IEMDT however shows leastsimilarities of 37 (Figure 2) In the 3-dimensional helicalfolding of the homologous proteins IEMDT shows twenty-one 120572-helices twelve 120573 strands and two n while SPLTB andSTT have two 120572-helices and five 120573 strands (Figure 3) Thisfurther removes IEMDT as a strong structural prediction forin pfmdr1

However it has been reported that typhoid toxin (STT)exhibits strong selectivity for Neu5Ac-terminated glycanswhich is predominantly expressed in human cells overNeu5Gc-terminated glycans predominantly expressed bymost othermammalsThis toxin bound a diverse groupof sia-lylated glycans with preferential binding to termini with theconsensus sequence Neu5Ac1205722-3Gal1205731-31205731-4GlcGlcNAcTherefore the exquisite binding selectivity of typhoid toxinfor glycans predominantly expressed in human cells providesan explanation for the inability of S typhi to cause typhoidfever in some nonpermissive species like chimpanzees Thisis consistent with previous results on crystal structure of pltb(SPLTB) and crystal structure of gd2 bound pltb which showsimilar structural conformation with pfmdr1 gene isolated inthis study [68] Since crystal structure of pltb and typhoidtoxin share similar structural conformation with the isolatedpfmdr1 gene in this study this could also be the reason whyP falciparum causes malaria only in humans Conclusivelythis compound (Neu5Ac1205722-3Gal1205731-31205731-4GlcGlcNAc) cantheoretically bind and change the functional integrity of thepfmdr1 protein thus providing a newwindow for drug target

Conflicts of Interest

The authors declare that no conflicts of interest exist regard-ing the publication of the manuscript

Acknowledgments

The authors would like to thank Professors Oyekanmi Nashand Mrs Hadiza Abdulrasheed of National BiotechnologyDevelopment Agency (NABDA) Abuja Nigeria for thevaluable support provided during the molecular study

References

[1] C O Agomo W A Oyibo C Sutherland R Hallet and MOguike ldquoAssessment of markers of antimalarial drug resistancein plasmodium falciparum isolates from pregnant women inLagos Nigeriardquo PLoS ONE vol 11 no 1 Article ID e01469082016

[2] World Health Organization ldquoGlobal technical Strategy formalaria elimination in the in endemic countries (2016ndash2030)rdquoWorld Health Organization Malaria Report World HealthOrganization Manila Philippines 2016

[3] J Tarning ldquoTreatment of malaria in pregnancyrdquo The NewEngland Journal of Medicine vol 374 no 10 pp 981-982 2016

[4] H A Antony S Das S C Parija and S Padhi ldquoSequenceanalysis of pfcrt and pfmdr1 genes and its association withchloroquine resistance in Southeast Indian Plasmodium falci-parum isolatesrdquo Genomics Data vol 8 pp 85ndash90 2016

[5] B Lawal O Shittu A Kabiru et al ldquoPotential antimalarialsfromAfrican natural products a reviewrdquo Journal of InterculturalEthnopharmacology vol 4 no 4 p 318 2015

[6] R McGready S J Lee J Wiladphaingern et al ldquoAdverse effectsof falciparum and vivax malaria and the safety of antimalarialtreatment in early pregnancy a population-based studyrdquo TheLancet Infectious Diseases vol 12 no 5 pp 388ndash396 2012

[7] P C Bhattacharyya and M Nayak ldquoMalaria in pregnancyrdquoMedicine Update vol 21 pp 475ndash478 2011


[8] J A Rowe J M Moulds C I Newbold and L H Miller ldquoPfalciparum rosetting mediated by a parasite-variant erythrocytemembrane protein and complement-receptor 1rdquo Nature vol388 no 6639 pp 292ndash295 1997

[9] A Barragan P G Kremsner M Wahlgren and J CarlsonldquoBlood group lsquoArsquo antigen is a coreceptor in Plasmodium falci-parum rosettingrdquo Infection and Immunity vol 68 no 5 pp2971ndash2975 2000

[10] F N Baliraine and P J Rosenthal ldquoProlonged selection ofpfmdr1 polymorphisms after treatment of falciparum malariawith artemether-lumefantrine in UgandardquoThe Journal of Infec-tious Diseases vol 204 no 7 pp 1120ndash1124 2011

[11] V Baraka H Tinto I Valea et al ldquoIn vivo selection of Plas-modium falciparumPfcrt and Pfmdr1 variants by artemether-lumefantrine and dihydroartemisinin-piperaquine in BurkinaFasordquoAntimicrobial Agents and Chemotherapy vol 59 no 1 pp734ndash737 2015

[12] A F Some Y Y Sere C Dokomajilar et al ldquoSelection of knownPlasmodium falciparum resistance-mediating polymorphismsby artemether-lumefantrine and amodiaquine-sulfadoxine-pyrimethamine but not dihydroartemisinin-piperaquine inBurkina Fasordquo Antimicrobial Agents and Chemotherapy vol 54no 5 pp 1949ndash1954 2010

[13] G S Humphreys I Merinopoulos J Ahmed et al ldquoAmodi-aquine and artemether-lumefantrine select distinct alleles ofthe Plasmodium falciparum mdr1 gene in Tanzanian childrentreated for uncomplicated malariardquo Antimicrobial Agents andChemotherapy vol 51 no 3 pp 991ndash997 2007

[14] C Fancony D Gamboa Y Sebastiao et al ldquoVarious pfcrt andpfmdr1 genotypes of Plasmodium falciparum cocirculate withP malariae P ovale spp and P vivax in Northern AngolardquoAntimicrobial Agents and Chemotherapy vol 56 no 10 pp5271ndash5277 2012

[15] Federal Ministry of Health National Guidelines and Strategiesfor Malaria Prevention and Control during Pregnancy FederalMinistry of Health Abuja Nigeria 2005

[16] I Simon-Oke O Afolabi and A Itansanmi ldquoPrevalence ofABOblood groups and its relationshipwithmalaria parasitemiaamong students of federal university of technology AkureOndo Staterdquo International Journal of Tropical Disease amp Healthvol 17 no 1 pp 1ndash8 2016

[17] M CheesbroughDistrict Laboratory Practice in Tropical Coun-tries Cambridge University Press Cambridge UK 2nd edition2005

[18] J V Dacie and S M Lewis Practical Haematology ChurchillLivinstone London UK 7th edition 1995

[19] C Mkandala ldquoThe effect of malaria in pregnancy on maternalanaemia and birth weight in rural Malawirdquo American Journal ofTropical Medicine and Parasitology vol 98 no 5 pp 213ndash2212003

[20] OG Raimi andC PKanu ldquoTheprevalence ofmalaria infectionin pregnant women living in a suburb of LagosrdquoNigeriaAfricanJournal of Biochemistry Research vol 4 no 10 pp 243ndash2452010

[21] O A Adefioye O A eyeba W O Hassan and O A OyeniranldquoPrevalence of malaria parasite infection among pregnantwomen in Osogbo Southwest Nigeriardquo American-EurasianJournal of Scientific Research vol 2 no 1 pp 43ndash45 2007

[22] I Omalu I Olayemi C Otuu et al ldquoEntomological andparasitological indices of malaria transmission inMinna NigerState North Central Nigeriardquo Advances in Research vol 3 no2 pp 181ndash188 2015

[23] I A A EjimaM A Yakub I K Olayemi and S O AbolarmwaldquoMalaria in pregnancy inminnametropolisminna Niger StateNigeriardquo Research Journal of Medical Sciences vol 7 no 4 pp110ndash117 2013

[24] I Georgian O Matthew and N I Nte ldquoPrevalence and effectof malaria in pregnancy among antenatal women in EbonyiState Nigeriardquo International Research Journal of Public andEnvironmental Health vol 4 no 8 pp 177ndash183 2017

[25] C Gamble J P Ekwaru and F O ter Kuile ldquoInsecticide-treatednets for preventing malaria in pregnancyrdquo Cochrane Databaseof Systematic Reviews vol 19 no 2 Article ID CD003755 2006

[26] T P Eisele D A Larsen P A Anglewicz et al ldquoMalariaprevention in pregnancy birthweight and neonatal mortality ameta-analysis of 32 national cross-sectional datasets in AfricardquoThe Lancet Infectious Diseases vol 12 no 12 pp 942ndash949 2012

[27] O Ayanda ldquoRelative abundance of adult female anophelinesmosquitoes in Ugah Nasarawa State Nigeriardquo Journal of Par-asitology and Vector Biology vol 1 no 1 pp 005ndash008 2009

[28] N Minakawa G Sonye M Mogi A Githeko and G Yan ldquoTheeffects of climatic factors on the distribution and abundance ofmalaria vectors in Kenyardquo Journal of Medical Entomology vol39 no 6 pp 833ndash841 2002

[29] O A Idowu C F Mafiana and S Dapo ldquoMalaria among preg-nant women in Abeokuta Nigeriardquo Tanzania Health ResearchBulletin vol 8 no 1 pp 28ndash31 2006

[30] B J Brabin ldquoAn analysis of malaria in pregnancy in AfricardquoBulletin of the World Health Organization vol 61 no 6 pp1005ndash1016 1983

[31] R U Allesandro and B J Langerock ldquoThe Risks and severity ofmalaria in pregnant womenrdquo Applied Field Research inMalariaReports TDRFIELDMAL1 WHO Geneva Switzerland 1998

[32] S N Obianumba Prevalence of malaria among pregnant womenattending antenatal clinic and knowledge and attitude of care-givers to malaria diagnosis In Ozubulu Anambra State [MSthesis] Nnamdi Azikiwe University 2012

[33] A P Kourtis J S Read and D J Jamieson ldquoPregnancy andinfectionrdquo The New England Journal of Medicine vol 370 pp2211ndash2218 2014

[34] D Muzzio M Zygmunt and F Jensen ldquoThe role of pregnancy-associated hormones in the development and function ofregulatory B cellsrdquo Frontiers in Endocrinology vol 5 article 392014

[35] G Mor and I Cardenas ldquoThe immune system in preg-nancy a unique complexityrdquo American Journal of ReproductiveImmunology vol 63 no 6 pp 425ndash433 2010

[36] I C J Omalu C Mgbemena A Mgbemena et al ldquoPrevalenceof congenital malaria inMinna North Central Nigeriardquo Journalof Tropical Medicine vol 2012 Article ID 274142 pp 1ndash5 2012

[37] C Falade O Mokuolu H Okafor et al ldquoEpidemiology ofcongenital malaria in Nigeria a multi-centre studyrdquo TropicalMedicine amp International Health vol 12 no 11 pp 1279ndash12872007

[38] P O Obiajunwa J A Owa and O O Adeodu ldquoPrevalenceof congenital malaria in Ile-Ife Nigeriardquo Journal of TropicalPediatrics vol 51 no 4 pp 219ndash222 2005

[39] A D Ekanem M U Anah and J J Udo ldquoThe prevalence ofcongenital malaria among neonates with suspected sepsis inCalabar NigeriardquoTropical Doctor vol 38 no 2 pp 73ndash76 2008

[40] M I Msellem A Martensson G Rotllant et al ldquoInfluence ofrapid malaria diagnostic tests on treatment and health outcomein fever patients Zanzibarmdasha crossover validation studyrdquo PLoSMedicine vol 6 no 4 Article ID e1000070 2009


[41] Ministry of Health andMedical Services Solomon Islands 2009Malaria Treatment Protocol Ministry of Health and MedicalServices Honiara Solomon Islands 2009

[42] O Ajumobi K Sabitu P Nguku et al ldquoPerformance of anHRP-2 rapid diagnostic test in nigerian children less than 5 years ofagerdquo The American Journal of Tropical Medicine and Hygienevol 92 no 4 pp 828ndash833 2015

[43] H Hopkins L Bebell W Kambale C Dokomajilar P JRosenthal and G Dorsey ldquoRapid diagnostic tests for malaria atsites of varying transmission intensity in Ugandardquo The Journalof Infectious Diseases vol 197 no 4 pp 510ndash518 2008

[44] E Nicastri N Bevilacqua M S Schepisi et al ldquoAccuracy ofmalaria diagnosis by microscopy rapid diagnostic test andPCR methods and evidence of antimalarial Overprescriptionin non-severe febrile patients in two Tanzanian hospitalsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 80 no5 pp 712ndash717 2009

[45] E E Ayogu Evaluation of malaria diagnostic methods efficacyof artemether-lumefantrine therapy and genetic determinants ofPlasmodium falciparum resistance [PhD thesis] University ofNigeria 2015

[46] M P Grobusch U Alpermann S Schwenke T Jelinek and DC Warhurst ldquoFalse-positive rapid tests for malaria in patientswith rheumatoid factorrdquo The Lancet vol 353 no 9149 p 2971999

[47] A H Moody and P L Chiodini ldquoNon-microscopic methodfor malaria diagnosis using OptiMAL IT a second-generationdipstick for malaria pLDH antigen detectionrdquo British Journal ofBiomedical Science vol 59 no 4 pp 228ndash231 2002

[48] J-H Lee J W Jang C H Cho et al ldquoFalse-positive results forrapid diagnostic tests for malaria in patients with rheumatoidfactorrdquo Journal of ClinicalMicrobiology vol 52 no 10 pp 3784ndash3787 2014

[49] Deepa V A Alwar K Rameshkumar and C Ross ldquoABO bloodgroups and malaria related clinical outcomerdquo Journal of VectorBorne Diseases vol 48 no 1 pp 7ndash11 2011

[50] O Kolawole O Ozokonkwo and O Mokuolu ldquoPrevalenceof plasmodium falciparum malaria among patients attendinguniversity of Ilorin teaching hospital Ilorin Nigeriardquo AnimalResearch International vol 11 no 3 pp 2063ndash2070 2014

[51] S E Amala and C P Nwibani ldquoMalaria in children itsassociation with abo blood group and haemoglobin genotyperdquoInternational Journal of Development Research vol 5 article 11pp 5958ndash5962 2015

[52] O I Leticia O E Ifeanyi C S Gideon E Queen and OK Chinedum ldquoDetermination of the haemoglobin genotypeand ABO blood group pattern of some students of Imo StateUniversity Owerri Nigeriardquo International Journal of CurrentReaserch and Academic Review vol 3 article 1 pp 20ndash27 2015

[53] A J Esan ldquoAssessment of haemoglobin variants in malariainfected individuals using haematological parametersrdquo Interna-tional Journal of Hematological Disorders vol 2 article 1 pp 4ndash9 2015

[54] R Bamou and S L Sevidzem ldquoABORhesus blood group sys-tems and malaria prevalence among students of the Universityof Dschang Cameroonrdquo Microwave Journal vol 7 article 42016

[55] H Tadesse and K Tadesse ldquoAssessing the association of severemalaria infection and ABO blood groups in northwesternEthiopiardquo Journal of Vector Borne Diseases vol 50 no 4 pp292ndash296 2013

[56] E Ito A Egwunyenga and J Ake ldquoPrevalence of malaria andhuman blood factors among patients in Ethiope East DeltaState Nigeriardquo International Journal ofMedicine and BiomedicalResearch vol 3 no 3 pp 191ndash201 2014

[57] M J Friedman ldquoErythrocytic mechanism of sickle cell resis-tance to malariardquo Proceedings of the National Acadamy ofSciences of the United States of America vol 75 no 4 pp 1994ndash1997 1978

[58] S O Ogbodo U I Nwagha A N C Okaka S C OgenyiR O Okoko and T U Nwagha ldquoMalaria parasitaemia amongpregnant women in a rural community Of eastern Nigerianeed for combined measuresrdquo Nigerian Journal of PhysiologicalSciences vol 24 no 2 pp 95ndash100 2009

[59] O Ogunbode ldquoManagement of anaemia in pregnancyrdquo NigeriaMedical Practice vol 8 pp 105ndash107 1984

[60] A A Akinbami S O Ajibola K A Rabiu et al ldquoHematologicalprofile of normal pregnant women in Lagos Nigeriardquo Interna-tional Journal of Womenrsquos Health vol 5 no 1 pp 227ndash232 2013

[61] E A Azab ldquoHaematological parameters in pregnant womenattended antenatal care at sabratha teaching hospital in North-west Libyardquo American Journal of Laboratory Medicine vol 2no 4 p 60 2017

[62] M Desai F O ter Kuile and F Nosten ldquoEpidemiology andburden of malaria in pregnancyrdquoThe Lancet Infectious Diseasesvol 7 no 2 pp 93ndash104 2007

[63] P E Ferreira G Holmgren M I Veiga P Uhlen A Kanekoand J P Gil ldquoPfMDR1 Mechanisms of transport modulationby functional polymorphismsrdquo PLoS ONE vol 6 no 9 ArticleID e23875 2011

[64] G M Boratyn A A Schaffer R Agarwala S F Altschul DJ Lipman and T L Madden ldquoDomain enhanced lookup timeaccelerated BLASTrdquo Biology Direct vol 7 article no 12 2012

[65] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[66] C Notredame D G Higgins and J Heringa ldquoT-coffee a novelmethod for fast and accurate multiple sequence alignmentrdquoJournal of Molecular Biology vol 302 no 1 pp 205ndash217 2000

[67] N Schormann B Pal O Senkovich et al ldquoCrystal structureof Trypanosoma cruzi pteridine reductase 2 in complex with asubstrate and an inhibitorrdquo Journal of Structural Biology vol 152no 1 pp 64ndash75 2005

[68] J Song X Gao and J E Galan ldquoConferring virulence structureand function of the chimeric A2B5 typhoid toxinrdquo Nature vol499 no 7458 pp 350ndash354 2013

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


main adult risk group for malaria [6] Pregnant women areparticularly susceptible to severe malaria and have a greaterrisk of antimalaria resistance due to loss of immunity duringpregnancy thus placing them at high risk of maternal andinfant morbidity and mortality abortion premature labourand stillbirth [7]

Genetic markers such as hemoglobin genotypes andblood groups have been associated with malaria but havebeen implicated with rosette formation and cytoadhesion [8]Variations in gene encoding functional glycotransferase havebeen associated with protection from P falciparum malariaAlso trisaccharide of ldquoArdquo and ldquoBrdquo blood group is presumedto act as receptor and function as an important factor forrosetting However the RBC of group O does not expresstrisaccharide blood types [9]

Genetic variations in pfmdr1 gene have played vital rolein malaria chemotherapy Mutations (N86Y Y184F S1034CN1042D and D1246Y) in the pfmdr1 gene have been asso-ciated with resistance to multiple antimalarial drugs such asquininemefloquine halofantrine artemisinin lumefantrineCQ and amodiaquine [10ndash13] However mutations in thisgene have been associated with geographic areas [14] andthere is a paucity of information on antimalarial drug resis-tance markers in Plasmodium falciparum infected pregnantwomen in Northern Nigeria Therefore it becomes relevantto determine the molecular marker of resistance in populaceof this region Also understanding the relationship betweenpfmdr1 amino acid sequence and its three-dimensional struc-tures will assist in predicting the chemical ligandsinhibitorthat can bind and change the functional integrity of theproteins This will provide information that is crucial indesigning an alternative and effective antimalarial drug thusproviding a new window for malaria drug target

2 Materials and Methods

21 Study Area This study was carried out in Minna NigerState located in the tropical zone of North Central NigeriaMinna the capital of Niger State Nigeria is located withinlongitude 6∘331015840E and latitude 9∘371015840N covering a land areaof 88 km2 with a population of 12 million (2006 PopulationCensus) Minna has a tropical climate with mean annualtemperature relative humidity and rainfall of 3020∘ 6100and 133400 cm respectively The climate presents two dis-tinct seasons a rainy season (AprilndashOctober) and dry season(NovemberndashMarch)

22 Inclusion andExclusionCriteria Allwomen enrolled hadearlier received intermittent preventive treatments (IPTp)with sulphadoxine-pyrimethamine (SP) as per current prac-tice The eligibility factor was set as women presentingwith gestational age 4 weeks and above to ensure adequatecoverage and data control Those who were not residentin the study area visitors and temporary residents whowere not officially registered for antenatal were not eligiblefor enrolment Other exclusion criteria included history ofadverse drug reaction or severe disease (such as hepatitisjaundice tuberculosis and obvious AIDS symptoms)

23 Ethical Considerations This study was approved by theEthical Committee of the Niger State Ministry of HealthMinna Nigeria Oral consent was obtained from the par-ticipant prior to inclusion in the study and before samplecollection All procedures were performed according to theguidelines for human experimentations in clinical researchas stated by the Federal Ministry of Health of Nigeria

24 Experimental Subjects and Sample Collection A total of250 women that attended antenatal care during the periodof study (December 2016) were recruited Each pregnantwoman was finger pricked using a sterile lancet to obtaincapillary blood for a rapid diagnostic test (RDT) for malariaSafety procedures were adopted in the collection of finger-prick blood samples by swabbing the area to be sampled with70 alcohol and allowing it to dry before collection Twomilliliters (2mL) of blood was then drawn (venipuncture)from malaria positive sample with a sterile disposable

25 Parasite Detection RDT based P falciparum specifichistidine-rich protein-2 (Bioline USA) was performedaccording to the manufacturerrsquos instructions to detect para-sites as recommended by the National Malaria Programme[15]

26 ABO Blood Grouping and Hemoglobin Genotyping TheABO blood group of each subject was determined using cellgrouping antisera (A B and D) as described by Simon-Okeet al [16] while the hemoglobin genotype separation wascarried out using cellulose acetate electrophoresis method asdescribed by Cheesbrough [17]

27 Hematological Parameters The hematological parame-ters hemoglobin (Hb) Packed Cell Volume (PCV) RedBlood Cells (RBC) White Blood Cells (WBC) and plateletscount (PLT) were determined using the automated hema-tologic analyzer SYSMEX KX21 a product of SYSMEXCorporation Japan as described by Dacie and Lewis [18]

28 Molecular Studies

281 DNA Extraction The genomic DNA was extractedfrom 2ml of infected blood using QIAmp DNA Mini Kit(Qiagen Hilden Germany) according to the manufacturerrsquosinstructions and subsequently stored at minus20∘C until use Thepresence of DNA was ascertained by subjecting it to 1agarose gel electrophoresis (DNA GelRed) and the purityof DNA was checked spectrophotometrically by calculatingthe A260A280 ratio

282 Amplification of pfmdr1 Gene Amplification of thepfmdr1 gene was performed on two fragments using poly-merase chain reaction (PCR) the first fragment containedcodons 86 and 184 the second fragment contained codons1034 and 1042 The primers were designed on the basis ofthe Plasmodium falciparum 3D7multidrug resistance protein(pfmdr1) partial mRNA sequence (GenBank accession noXM 0013517511) The reaction primers and conditions for

















3 Results








































4 Discussion






Table7PD

BSum

database

inform

ationof

pfmdr1g

eneh

omologou

sprotein

sequ

enceSequenceGTD

YFCN

LIEK

AID

YKNKG

QKR

RIIV

NAALW

GFS

QSA

QLF

INSFAY

WFG

SXLIKR

GTIL-

VDDFM

KXX

Sequ

ence

leng

th67resid

ues

PDB

code

Mod

elLeng

th-ta

geidentity

aa

overlap

119911-score

Ligand

sProteinname

13w

mg(A)

X-ray240

A589

273

441398

DMU

TRS

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


incomplex

with

acyclic

peptideinh

ibito

raC

AP

23w

mf(A

)X-

ray260

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


3IEMDT(A)

X-ray275

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter

44rhs(A

)X-

ray19

2A114

317

411269

ACTSIA-

SIA-

GAL

Crystalstructure

ofGD2

boun

dpltb

5SP

LTB(A)

X-ray208

A114

317

411269

ACT

Crystalstructure

ofpltb

6ST

T(A)

X-ray239

A114

317

411269

GOL

Structureo

ftypho

idtoxin

75luq

(A)

X-ray430

A3725

348

461116

Crystalstructure

ofhu

man

DNA-

depend

entp

rotein

kinase

catalytic

subu

nit

(DNA-

PKcs)


BAD AVG GOOD



cons



cons

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

lowast

lowast

lowast lowast

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons



cons


IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT


cons





TT TT





TT

TT


TT TT TT TTT TT


TT TT

TT


TT


TT TT


MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600



1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2



(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































3 Results








































4 Discussion






Table7PD

BSum

database

inform

ationof

pfmdr1g

eneh

omologou

sprotein

sequ

enceSequenceGTD

YFCN

LIEK

AID

YKNKG

QKR

RIIV

NAALW

GFS

QSA

QLF

INSFAY

WFG

SXLIKR

GTIL-

VDDFM

KXX

Sequ

ence

leng

th67resid

ues

PDB

code

Mod

elLeng

th-ta

geidentity

aa

overlap

119911-score

Ligand

sProteinname

13w

mg(A)

X-ray240

A589

273

441398

DMU

TRS

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


incomplex

with

acyclic

peptideinh

ibito

raC

AP

23w

mf(A

)X-

ray260

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


3IEMDT(A)

X-ray275

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter

44rhs(A

)X-

ray19

2A114

317

411269

ACTSIA-

SIA-

GAL

Crystalstructure

ofGD2

boun

dpltb

5SP

LTB(A)

X-ray208

A114

317

411269

ACT

Crystalstructure

ofpltb

6ST

T(A)

X-ray239

A114

317

411269

GOL

Structureo

ftypho

idtoxin

75luq

(A)

X-ray430

A3725

348

461116

Crystalstructure

ofhu

man

DNA-

depend

entp

rotein

kinase

catalytic

subu

nit

(DNA-

PKcs)


BAD AVG GOOD



cons



cons

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

lowast

lowast

lowast lowast

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons



cons


IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT


cons





TT TT





TT

TT


TT TT TT TTT TT


TT TT

TT


TT


TT TT


MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600



1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2



(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























































4 Discussion






Table7PD

BSum

database

inform

ationof

pfmdr1g

eneh

omologou

sprotein

sequ

enceSequenceGTD

YFCN

LIEK

AID

YKNKG

QKR

RIIV

NAALW

GFS

QSA

QLF

INSFAY

WFG

SXLIKR

GTIL-

VDDFM

KXX

Sequ

ence

leng

th67resid

ues

PDB

code

Mod

elLeng

th-ta

geidentity

aa

overlap

119911-score

Ligand

sProteinname

13w

mg(A)

X-ray240

A589

273

441398

DMU

TRS

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


incomplex

with

acyclic

peptideinh

ibito

raC

AP

23w

mf(A

)X-

ray260

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


3IEMDT(A)

X-ray275

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter

44rhs(A

)X-

ray19

2A114

317

411269

ACTSIA-

SIA-

GAL

Crystalstructure

ofGD2

boun

dpltb

5SP

LTB(A)

X-ray208

A114

317

411269

ACT

Crystalstructure

ofpltb

6ST

T(A)

X-ray239

A114

317

411269

GOL

Structureo

ftypho

idtoxin

75luq

(A)

X-ray430

A3725

348

461116

Crystalstructure

ofhu

man

DNA-

depend

entp

rotein

kinase

catalytic

subu

nit

(DNA-

PKcs)


BAD AVG GOOD



cons



cons

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

lowast

lowast

lowast lowast

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons



cons


IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT


cons





TT TT





TT

TT


TT TT TT TTT TT


TT TT

TT


TT


TT TT


MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600



1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2



(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Table7PD

BSum

database

inform

ationof

pfmdr1g

eneh

omologou

sprotein

sequ

enceSequenceGTD

YFCN

LIEK

AID

YKNKG

QKR

RIIV

NAALW

GFS

QSA

QLF

INSFAY

WFG

SXLIKR

GTIL-

VDDFM

KXX

Sequ

ence

leng

th67resid

ues

PDB

code

Mod

elLeng

th-ta

geidentity

aa

overlap

119911-score

Ligand

sProteinname

13w

mg(A)

X-ray240

A589

273

441398

DMU

TRS

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


incomplex

with

acyclic

peptideinh

ibito

raC

AP

23w

mf(A

)X-

ray260

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter


3IEMDT(A)

X-ray275

A588

273

441398

DMU

Crystalstructure

ofan

inward-facing

eukaryotic

ABC

multid

rugtransporter

44rhs(A

)X-

ray19

2A114

317

411269

ACTSIA-

SIA-

GAL

Crystalstructure

ofGD2

boun

dpltb

5SP

LTB(A)

X-ray208

A114

317

411269

ACT

Crystalstructure

ofpltb

6ST

T(A)

X-ray239

A114

317

411269

GOL

Structureo

ftypho

idtoxin

75luq

(A)

X-ray430

A3725

348

461116

Crystalstructure

ofhu

man

DNA-

depend

entp

rotein

kinase

catalytic

subu

nit

(DNA-

PKcs)


BAD AVG GOOD



cons



cons

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

lowast

lowast

lowast lowast

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons



cons


IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT


cons





TT TT





TT

TT


TT TT TT TTT TT


TT TT

TT


TT


TT TT


MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600



1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2



(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















BAD AVG GOOD



cons



cons

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------


cons

PFMDR1 ------------------------------------------------------------------------SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons

lowast

lowast

lowast lowast

PFMDR1 ------------------------------------------------------------------------

SPLTB ------------------------------------------------------------------------STT ------------------------------------------------------------------------


cons



cons


IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT

IEMDT


cons





TT TT





TT

TT


TT TT TT TTT TT


TT TT

TT


TT


TT TT


MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600



1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2



(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















TT TT





TT

TT


TT TT TT TTT TT


TT TT

TT


TT


TT TT


MSAAGSENLYFQ

IEMDT1 1 0 2 0 3 0 4 0 5 0 6 0

IEMDTSTTSPLTBSPLTB

IEMDT7 0 8 0 9 0 1 0 0 1 1 0 1 2 0

IEMDTSTTSPLTBSPLTB

IEMDT1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0

IEMDTSTTSPLTBSPLTB

IEMDT1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0

IEMDTSTTSPLTBSPLTB

IEMDT2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0

IEMDTSTTSPLTBSPLTB

IEMDT3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0

IEMDTSTTSPLTBSPLTB

IEMDT3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0

IEMDTSTTSPLTBSPLTB

IEMDT4 3 0 4 4 0 450 4 6 0 470 4 8 0

IEMDTSTTSPLTBSPLTB

IEMDT4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0

IEMDTSTTSPLTBSPLTB

IEMDT5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 600



1 2

43

6

5

7

98

10

11

13 141

1

12

15 16

1

1817

19

2

2120

1

2

1

4

3

532

6 7

8

4

10 11 12

9

5

1

1

2

2 2



(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b)

(c)

































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of







































Acknowledgments


References











































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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