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REVIEW ON THE USE OF GENE EXPRESSION DATA IN THE STUDY OF HUMAN MALARIA

DISEASE.

BY ONYIA, CHIADIBOBI E.SCHOOL OF BIOMEDICAL SCIENCES, CARDIFF METROPOLITAN UNIVERSITY,

UK.

MALARIA OVERVIEW

Malaria with symptoms like fever and headache is a human disease caused by Plasmodium falciparum and transmitted by female Anopheles mosquito. The disease affects over 400 million people worldwide, mainly Africa, Asia and South America killing nearly one million people per annum (Kritsiriwuthinan et al., 2011) with 91% of cases in Africa (WHO, 2012) (Bachmann et al., 2011; Volz et al., 2012).

Plasmodium falciparum undergoes cyclic developmental life cycle, where the oocysts produced by the fusion of the gametes develop into sporozoites in the mosquito mid-gut and transmitted to the human host during the mosquito blood meal. The matured sporozoites in the hepatocyte are released into the blood as merozoites (Boddey and Cowman, 2013) as shown in fig.1.

Fig. 1a: Malaria the fourth leading deadly disease in Africa (WHO, 2012) as indicated by the arrow.

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Fig. 1b: The life cycle of Plasmodium falciparum (McW Healthcare, 2008). Female Anopheles mosquito on feeding with on human host blood transmits sporozoite to the hepatocyte, which develop and are released to invade the erythrocyte as merozoite. It is the initiates the production of pfemp1 that mediate malaria infection.

The merozoites released from the hepatic region internalized the cell by bind to the glycophorin-A receptor complex (see fig 2ii) on the erythrocyte membrane and remolds the erythrocyte, by altering the erythrocytic normal gene expression (Zhang et al., 2014). The reason why the parasite chose to use the red blood cell, might be because of the presence of iron that help them mediate its transcription. The presence of haem-iron in the erythrocyte helped merozoite with its machinery to switch on its gene expression, where by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and other proteins are produced as they undergo various stages of development (namely; ring stage, trophozoite stage and schizont stage) inside the human red blood cell, (Boddey and Cowman, 2013; Zhang et al., 2014) as shown in fig 2i.

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Fig.2. (i) The asexual intraerythrocytic developmental cycle (IDC). From 0-8h (ring stage), 8-16h (early trophozoite), 16-24h (late trophozoite stage), 24-32h (early schizont), 32-40h (late schizont stage), 40-48h (assembly and release of PfEMP1 protein for infection) (ii) merozoite internalization via band-3-glycophorinA complex (Rai et al., 2014; PNAS, 2010)

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The PfEMP1 produced, binds to uninfected erythrocyte (rosetting) with its duffy binding like (DBL) region, while cysteine-rich interdomain region (CIDR) mediates binding to inter cellular adhesion molecule1 (ICAM-1) and cluster of differentiation 36 (CD36) receptors in the endothelial of the human cells (Duffy et al., 2002; Pasternak and Dzikowski, 2009). The rosetting prevents the cell from being destroyed by the splenic macrophage (Scherf et al., 2008; Boddey and Cowman, 2013). But this antigenic PfEMP1 is encoded by variant (var) gene.

Var gene family of about 60 genes is being grouped into three main upstream sequences (ups), based on their transcriptional orientation, as upsA, upsB and upsC (Witmer, 2011; Volz, 2012), whereby upstream sequence A (upsA) var genes and upsB genes are located in the two subtelomeric regions while upsC are centrally located within the telomeric region of the chromosome.

Kyes et al., (2007) stated that most var gene expression occur during the ring stage while Zhang et al., (2014) and Albrecht et al., 2011 on exploring var3 and var2 genes respectively observed that their transcription peak fall within late trophozoite stage. Further study conducted by Noble et al., (2013) on the antigenic switching of thirty-five different var genes and their implication for malaria disease, concluded that most var gene are expressed at the three stages of their intraerythrocytic developmental cycle. This provoked an initial debate on whether var gene location contributes to its gene expression efficiency.

But since it has been established that var genes encode for the main virulence antigenic PfEMP1, it is evident that var2 and var3 genes forms the bases of this review concerning malaria disease, based on the fact that they possessed duffy binding like (DBL) core domain within their PfEMP1 (see fig 11) which is the bases of malaria infection occurrence.

VAR GENES EXPRESSION METHODS FOR MALARIA DISEASE

For merozoite to cause malaria, PfEMP1 antigenic protein must be produced, but there are various research works conducted on var2 and var3 genes expression. Their work showed that these genes expresses either micro inhibitory RNA (miRNA) that does not produce PfEMP1 or mRNA which produces the main antigenic variant (PfEMP1), which gave rise to malaria disease in man (Pasternak and Dzikowski, 2009). In that essence, the severity of any var gene expression lies on its ability to produce PfEMP1.

Therefore, studies on var2 and var3 gene expressions have been determined using various methods like microarray method, northern blotting and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) (QPCR) method as described below.

Microarray method protocol: Due to microarray competency in accessing multiple genes at a time, this method was used to determine the likely gene expressions that are responsible for malaria infection and probably its treatment following these protocols:

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Infected and non-infected human volunteers and animal models were used, whereby total RNA were extracted from infected and non-infected erythrocyte using TRIzol reagent (Kritsiriwuthinan et al., 2011).

The RNA obtain were converted to mRNA poly (A) using oligo (dT) and then to cDNA using reverse transcriptase, for easy labeling. Each cDNA used were labelled using different fluorescent dye like (cyanine) Cy3, Cy5 monoreactive dye (Kritsiriwuthinan et al., 2011).

The labelled cDNA transcripts were transferred unto an affymetrix gene-chips micro-chip slide, wherein several oligo nucleotides have been (embedded) incorporated for hybridization to take place (Kulkarni et al., 2012). The cDNA differently labeled hybridized, with different colour indication as shown in fig 3.

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Fig.3 Microarray data analysis: Sample from human iRBC, reverse transcribed and labeled with cyanine dye. The labeled dyes then hybridized and visualize The result showed that the micro-chip that have already been embedded with oligo nucleotides hybridized with the appropriate complementary cDNA labelled strand and fluorescence, wherein colored black showed un-infected (i.e. no expression and no hybridization), green showed moderate expression and red showed high expression (Kulkarni et al., 2012).

Northern blotting protocol: This method was used to isolate the specific RNAs from the genes of interest for more detailed study. Kyes et al., (2007) identified Plasmodium falciparum var gene expression using northern blotting method; firstly RNAs were obtained using Trizol reagent to lyse the parasitized synchronized red blood cell (pRBC) at ring, trophozoite and schizont stages of parasite cycle.

Secondly the RNAs extracted from pRBC were hybridized with α-32P-dATP mega prime probe and visualise using agarose gel that containing ethidium bromide (Kyes et al., 2007), knowing that the band intensity is proportional to the amount of target mRNA present.

This method proved that different quantities of mRNAs were expressed at difference stages of the parasite intraerythrocytic developmental cycle.

RT-qPCR (Real-time PCR) protocol: Albrecht et al., (2011); Bachmann et al., (2011); Noble et al., (2013) and Zhang et al., (2014) on their various var genes exploration, used RT-qPCR method for var gene expression. RT-qPCR method measures PCR amplicon as it occurs, so that the concentration of RNAs will be determined at its linear phase (Applied Biosystem, 2010).

Sample collection for successful RT-qPCR protocol: Experimental sample, dNTP-mix, forward and reverse primer, Taq polymerase, buffer, nuclease free water, reverse transcriptase and real-time PCR which contains a fluorescent reporter molecule, TaqMan probe or SYBR Green dye were collected(Applied Biosystem, 2010).RNA isolation, confirmation and reverse transcription procedure: RNAs isolate were obtained firstly by lysing the cell and by treating them with DNase1 to remove the whole genomic DNA (gDNA) present and seryl-tRNA synthetase were used to confirm the absence of gDNA which would have caused spurious result (false positive result) (Bachmann et al., 2011). The purified polyadenylated mRNA present were reversed transcribed to double stranded cDNA by adding oligo (dT) primer (see fig.4). Rename H were also added to

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remove the RNA strand of the double stranded cDNA leaving only single stranded DNA as the template cDNA ready for qPCR assay (Sellner an d Turbett, 1998).

Fig.4 Prerequisite for determination of mRNA level: The mRNAs are being reverse transcribed to cDNA that is more stable for PCR amplification (Applied Biosystem, 2010)

Primer design for RT-qPCR method: Good gene specific primers were designed following the standard method of obtaining 40-60% of GC content, melting temperature of 58-600C , base length of 18-25 and targeting of the 3’ untranslated region (Holland et al., no-date).

SYBR Green probe for RT-qPCR quantification: Albrecht et al., (2011) and Bachmann et al., (2011) used Power SYBR Green Master mix as their fluorescent dye. As the primers elongate the single stranded cDNA sequence, SYBR Green being a non-specific probe binds to the double stranded cDNA to fluorescence (see fig. 7b) (Thomas, 2014). Knowing that SYBR Green is non-specific, melting analysis was used to confirm the PCR product specificity (Noble et al., 2013) should in-case primers hybridized.

TaqMan probe for RT-qPCR quantification: Others used TaqMan probe (hydrolysis probe) which is sensitive, specific and are capable of quantitating the target PCR product. TaqMan probe mechanism, shows that the experimental genes (cDNA of interest) at 950C PCR temperature were denatured and the specific hybridize probe binds (anneals) to the transcript using its 5’ to 3’ exonuclease (see fig. 5) (Sellner et al., 1998). The probe was designed by placing reporter fluorescent and a quencher at very close proximity so that the reporter will not be able to fluorescence except when disassociated from the quencher, as shown in fig.5 and 7a.

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Figure 5: TaqMan Gene Expression Assay reaction steps (Applied Biosystem, 2012). DNA template denatures allowing probe and primer to anneal. Primer binding mediate Taq polymerase to elongate, separating the reporter from quencher allowing reporter to fluorescence.

Threshold line Exponential stage Stationary phase

Fig 6. RT-qPCR product visualization through graph display (Adopted from, Applied Biosystem, 2010), were the gene expression product is made visible when the emission crosses the threshold line. The higher the start template the faster it crosses the threshold line. This can be applicable to both TaqMan and SYBR Green probe only that TaqMan product will be more specific.

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a b

Fig. 7. (a) 5’ Nuclease (TaqMan Probe) Assay (b) SYBR Green Dye Assay

Here shows the differences between the use of TaqMan and SYBR Green probe. TaqMan has a specific oligo nucleotide with reporter dye and quencher and binds to a specific template, whereas SYBR Green probe binds to any double strand present making it nonspecific in that it can detect probe hybrid as count which is a false positive result (Applied Biosystem, 2010).

In probe designing it was made in such a way that the probe annealing temperature is 5-100C greater than the primer annealing temperature (Sellner et al., 1998) and the temperature variation encourages the probe to anneal first to the single stranded cDNA before the primer, because primer annealing initiates extension. If primer anneal and extent without the probe, photon will not be emitted.

Primer binding causes the Taq polymerase to commence extension of the transcript, which knocks-out the probe thereby separating the reporter from the quencher and the reporter emit photon that are detected by the fluorimeter (Sellner et al., 1998). As the cycle continues the emission increases, displaying a graph chat as shown in fig 6, creating an exponential phase that crosses the threshold line as the PCR product increases.

CRITICAL ANALYSIS ON VAR GENE EXPRESSED DATA

Considering the vast work conducted by many researchers in the area of Plasmodium falciparum var gene expression, their works reveal the likely causes and possible approaches to malaria disease, which is anchored on var gene expression and its protein sequestration as shown in fig 8.

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Fig. 8: Schematic flow chart of PfEMP1 key role in malaria pathogenicity Parasite infection on the erythrocyte expresses var genes which are translated into PfEMP1 transported by the MC to the infected red blood cell (iRBC) membrane. PfEMP1 binding to endothelial cells of the host, causes sequestration and cell rosetting which leads to malaria. [Intracellular adhesion molecule 1 (ICAM-1), Chondroitin sulphate A (CSA), Parasitophorous vacuole (PV), Maurer’s cleft (MC)] (Pasternak and Dzikowski, 2009)

Knowing that var gene encodes PfEMP1 that causes sequestration (binding) in an endothelial part of any part of the body which determines the severity of malaria. Kraemer and Smith, (2006) showed that the binding of PfEMP1 to ICAM-1 or chondroitin sulphate A (CSA) using DBL domain cause the most severe malaria infection. It is evident that ICAM-1 causes erythrocyte sequestration within the brain region which leads to cell auto-agglutination, apoptosis and probably death of the host (Siau et al., 2007). If the sequestration was unto CSA within the intervillous spaces of the placenta during pregnancy, it can also cause a severe pregnancy associated malaria (PAM) that might lead to premature delivery or severe anemia or even death of the mother and the fetus (Pasternak and Dzikowski, 2009). These dangers can be circumvented by interfering the var gene expression. Var2 and var3 gene are the gene that expresses the mRNA that encodes PfEMP1 within the most appropriate domain as shown in figs. 9a and 9b were found to up regulate its gene at DBLα1.7 which is the domain cassette 13 as shown in fig.10 . Goel et al., (2014) in their study on the most expressing var gene, observed that var2 expressed higher quantity of mRNA (see fig. 9a) which adherence to endothelial cell surface molecules, using CD36 and ICAM-1.

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Fig. 9a: Var gene expression. Var2 gene obtained from Plasmodium falciparum strain B, showing high gene expression product and invariably causes more severe malaria infection (Goel et al., 2014).

Further study on var gene expression analysis by Albrecht et al., (2011) observed that out of sixteen var gene family analyzed using RT-qPCR method only var2 gene were genuinely expressed as was shown in fig.9. This gives an impression that all var genes are not always on for expression based on its position on the telomere, but are switched on or off by a certain switching agent (ligand). Knowing that var2 gene is sub telomeric located, Albrecht et al., (2011) disproved previous claim made by Frank et al., (2007) and Peters et al., (2007) suggested that high gene expression product exhibited by some var genes was as a result of their location in the telomere, stressing that centrally located genes expresses’ more than the sub-telomeric gene as was claimed by some authors. This might be because some subtelomeric deletion of ~100 kb in chromosome 2 which resulted in significant reduction in the cytoadherence, as this region contains the knob-associated histidine rich protein (KAHRP) gene, which is crucial for the assembly of a rigid knob and a clustering of PfEMP1 in the knob (Goel et al., 2014).

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Fig. 9b. Relative var transcript levels of sixteen var genes using QPCR method with var2 gene as the gene expressed (Albrecht et al., 2011). This figure showed var2 gene as the only gene that expressed its gene in this cascade of genes.

Volz et al., (2012), on exploring cascade of var gene observed that var2 gene expression was great, but further discovered that a histone-3-lysine-4- methyltransferase (H3L4M) were required to maintain var gene in the active state during expression. This shows that inhibition H3L4M inhibits var2 gene expression (see fig. 13a).

Smith et al., 2001 showed that var gene expression of coding transcript region depends only on the ability of each gene sequence to properly express the core sequence domain (see fig.10a and 10b) that cut-across the intraerythrocytic developmental cycle (IDC) pathway. The core regions in the var genes if properly expressed makes antigenic protein, but if not expressed, brings miRNA transcript that cannot form proteins and can disintegrate so easily (Claessens et al., (2012). This might be the same reason why some detected transcript disintegrates with time, because of their inability to express the appropriate domain sequence. It is clear from this evidence that DBL exon expression produces antigenic protein and this also agreed with Scherf et al., (2008) which proved that in most var genes coding exons, the first exon encode for the PfEMP1 extracellular potion for binding while the second exon encode for the transmembrane and intracellular region of PfEMP1.

Fig.10a. PfEMP1 architecture of variants up-regulation: Each var gene has difference sequence of these core regions that must be up regulated for mRNA transcript to be made. [ATS=acidic terminal segment; CIDR= cysteine-rich interdomain region; DBL= Duffy binding-like domain; DC13=domain cassette 13; DC8= domain cassette 8; NTS= N-terminal segment] Var 2 and var3 are among the genes that transcribe the core domain for the production of virulent PfEMP1. (Smith et al., 2001). Expression of DBL1α and DBL2β mediate for more severe malaria infection.

Fig.10b: Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) conservation and polymorphism. (a) Duffy-binding-like domain (DBL) sequence conservation is significantly concentrated in ten semi conserved homology blocks (coloured pink and shown as the

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same size for simplicity) flanked by ten hypervariable blocks (blue). DBL domains display type-specific differences in length and molecular mass. Size variation is illustrated as the average length difference between DBLα and δ types. Distributed unequally among homology blocks are ten conserved cysteine residues (C1–C10) present both in PfEMP1 and erythrocyte-binding antigen (EBA) DBL domains. Additional type-specific cysteine residues are also present12. Crucial EBA DBL binding residues map between C4 and C7 (Ref. 53). (b) Cysteine-rich intracellular domain region (CIDR) domains can be divided into three areas (M1, M2 and M3) based on the Malayan Camp CIDR1, in which the minimal CD36 binding region (M2) has been defined. M1 and M2 areas with greater sequence conservation are indicated with bold black lines. The M3 area is extremely degenerate (indicated with green background) and frequently contains runs of charged residues. The general position of 13 conserved cysteine residues (C1–C13) are shown for CIDRα and β types. Additional typespecific cysteines are coloured orange (CIDRα) or green (CIDRβ). (c) Comparisons of CIDRα and β consensus motifs, with invariant cysteine (Smith et al., 2001).

Fig. 11 Var gene transcripts from patients with cerebral malaria and uncerebral malaria in Benin[White-bar =˂ 2%, light-gray-bar = ˂ 5% and mid-grey-bar =˂ 20%] the higher the % gene expressed the more severe the malaria and the more the core gene expressed. Since more mid-grey-bars were formed at the cerebral malaria samples section, this proved that expression of the core gene sequence took place (Bertin et al., (2013) for cerebral malaria to occur. The arrows spotted the core DBL expression which is the main reason for severe malaria.

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Fig12: Var gene expression quantification against apoptosis in malaria disease. Characteristics of the three nonapoptogenic (NA1–NA3) and the five apoptogenic (A1–A5) isolates were detailed and compared with strain 3D7. This shows that some var transcript might not be apoptogenic and cannot cause severe malaria disease. The genes with red and asterisk are genes that express most severe malaria infection and the arrow indicate var2 gene with high transcript in A1-A5 strains (Siau et al., 2007)

Zhang et al., (2014) on exploring the var gene sequence of Plasmodium falciparum, observed that var3 genes are found to conserved and encode only four domains which were identified to be N-terminal segment (NTS), Duffy binding-like (DBL), Transmembrane region (TM) and Acidic terminal segment (ATS). The inability to transcribe the right domain might be the reason why some var gene expression disintegrates. These claims were further clarified when Bertin et al., (2013) compared the cerebral malaria with the non-malaria person, which showed that core gene region when (expressed) transcribe produces more stable transcript as shown in fig.11, than when not properly expressed.

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Fig 13a: Prerequisites of nucleosome formation and contexts of histone chaperone activity. Histone chaperones may facilitate nucleosome formation by being involved in some or all of the processes I-VI indicated. Histones may be transferred between chaperones to complete all these processes in a regulated manner (Elsasser and Arcy, 2013).

The issue of proper gene switching is attributed to histone dimerization which mediate proper incorporation. The histone serves as chaperone that help mediate proper switching as was explained in figs. 13a and 13b.

Fig.13b: A proposed model for the regulation of var gene expression in the expression site Cytoplasm (Cy), nucleus (N), histone 3 (H3), (H2A) Histone 3 epigenetic modifications involved in var gene regulation were proposed. Var gene transcription is characterized by H3K4 trimethylation, H3K9 acetylation, and H2AZ for histone variant. Loss of histone variant H2AZ from the active var promoter causes silencing of var genes. After replication, canonical histones such as histone H2A and H3 are incorporated to the var promoter providing a window of opportunity for switching and silencing. Histone H2.AZ is deposited at the var promoter during ring stage and var gene silencing involves movement of the var gene locus out of the expression site. Deacetylation is likely mediated by SIR2 homologs and H3K9 trimethylation by a yet unknown HKMT (Volz et al., 2012).

PfEMP1 encoded by var genes, constitute the major parasite virulence factors that are made of a long coding exon (NTS, DBL, CIDR and TM) and a short ATS exon separated by an intron which is the domain architecture for malaria disease initiation (Zhang et al., 2014). There are two promoters within each gene sequence, one at the upstream open reading frame for mRNA expression and the other within the intron which produces a sterile transcript that regulates the expression (Pasternak and Dzikowski, 2009). The histone serves as a chaperone that initiates transcription.

There is likely speculation that if the ATS segment of the gene is blocked, the mRNA expression product will stop, and if the mRNAs are not expressed, PfEMP1 will not be formed thereby preventing malaria disease. It has also been established that different DBL and CIDR binding repeats functions differently, Kraemer and Smith, (2006) established that PfEMP1 binding on CD36 mediated by CIDR-1α causes mild malaria while PfEMP1 binding on ICAM-1 or CSA mediated by DBL-2β and DBL-ɛ respectively causes severe malaria. Almelli et al., (2014) knocked-out CIDR-1α gene segment prevented bind to CD36, proposing that this knock-out procedure can be of help in preventing malaria disease. The understanding of var gene expression has revealed some likely pathways or sequence that

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might be the target site for drugs and also epi-drug pathway that will help inhibit malaria menace if properly implemented.

VAR GENE EXPRESSION APPROACH TO MALARIA PREVENTION AND TREATMENT

The malaria disease approach through gene expression, has unfolded the genetic pathway to this disease. This explained how the genes encode for merozoite surface proteins (msp) within the hepatocyte egress (Boddey and Cowman, 2013; Gupta et al., 2013). Knowing that epigenetic regulations mechanisms are the hallmark of malaria infection on the platform of IDC transcriptional activity (Gupta et al., 2013).

Gupta et al., (2013) explored that most anti malaria drugs through epigeneticity, remodels the chromatin structure and prevent transcriptional activity. Using a mice model, Darkin-Rattray et al., (1996) discovered that apicidin (anti malaria) when administered orally inhibits histone deacetylase (HDAC) thereby distorting the normal remodeling initiation that mediate transcription, which invariably deregulate the IDC transcriptional cascade. This evidence on gene expression, paved way for the invention of apicidin that inhibits transcriptional initiation.

Usually anti-malaria drugs like chloroquine, mefloquine and sulfodoxine-pyrimethamine usually binds to haem-iron which down regulate gene expression by inhibiting the alpha-haematin formation, knowing that haem reduction down regulate gene expression (Kritsiriwuthinan et al., 2011; Mok et al., 2011). Down regulation of gene, makes the erythrocyte not to be sequestered and can be killed by macrophage. But some strains devised other pathway where by miRNA produced up regulates gene expression thereby rendering those drugs impotent. Based on this researcher ventured on the use of combine therapy which might be of help to the menace.

Artemisinin combination therapy (ACT) was produced with much prolonged parasite clearance-mean time (PCT) of 84 hours, and was recommended by the World Health Organization as anti-malaria drug of choice (Mok et al., 2011). Recently ACT resistant strain has occurred. The newly discovered ACT resistant parasites has a conserved exon that are expressed at schizont stage for which histone 4 are normally use in the progressive assembly. Therefore, more new antimalaria (epi-drugs) that may inhibit both HDAC and histone 4 and other chromatin remodeling complexes (Prado and Aguilera, 2005; Mok et al., 2011) will be of help in treating malaria.

Now, the knowledge of gene expression has enlightened us on the possible route the parasite follows in the course of its normal life cycle and the stages that interference can occur so as to inhibit malaria disease. It is now clear that var2, var3, when full expressed, give rise to PfEMP1 which serves as both variant antigen, parasite virulence factor (Pasternak and Dzikowski, 2009; Noble et al., 2013).

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Having known all these facts about malaria and its var gene expression, these evidences suggest that we can ameliorate the infection by either preventing human contact with female anopheles mosquitoes where the first phase of the parasites develops, or mimic the merozoites binding at glycophorin-A receptor to block the main parasitic merozoite from internalizing the cell or by developing more new combine therapy that can interfere and remodel various chromatin complexes at all IDC stages thereby incapacitating all transcriptional activities. Knowing that PfEMP1 is the main virulent protein, production of anti-malaria drugs that will serve as chaperone to misfold this protein at the post translational stage thereby producing pseudo-PfEMP1.

References

Applied Biosystem (2010). Available at: www.appliedbiosystem.com. Accessed date 28-10-2014.

Applied Biosystem (2012). Available at: www.appliedbiosystem.com. Accessed date 28-10-2014.

Albrecht, L., Moll, K., Blomqvist, K., Normark, J., Chen, Q., et al., (2011). Var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2. Malaria Journal, 10:17-25.

Almelli, T., Ndam, N.T., Ezimegnon, S., Alao, M.J., Ahouansou, C., et al., (2014). Cytoadherence phenotype of Plasmodium falciparum infected erythrocytes is associated with specific pfemp-1 expression in parasites from children with cerebral malaria. Malaria Journal, 13(1):333-342.

Bachmann, A., Predehl, S., May, J., Harder, S., Burchard, G.D., et al., (2011). Highly co-ordinated var gene expression and switching in clinical Plasmodium falciparum isolates from non-immune malaria patients. Cellular Microbiology, 13(9):1397-1409.

Bertin, G.I., Lavstsen, T., Guillonneau, F., Doritchamou, J., Wang, C.W., et al., (2013). Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin. PLOS ONE 8 (7): e68368.

Boddey, J.A., and Cowman, A.F., (2013). Plasmodium nesting: remarking the erythrocyte from the inside out. Annual Review of Microbiology, 67:243-269.

Bustin, S.A., (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4):611-622.

Claessens, A., Adams, Y., Ghumra, A., Lindergard, G., Buchan, C.C., et al., (2012). A subset of group A-like var genes encodes the malaria parasite ligands for binding a human brain endothelial cell. Proceedings of the National Academy of Science of USA, 109(26):1772-1781.

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Darkin-Rattray, S.J., Gurnett, A.M., Myers, R.W., Dulski, P.M., Crumley, T.M., et al., (1996). Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase. Proceeding of the National Academy of Science, USA, 93:13143-13147.

Duffy, M.F., Brown, G.V., Basuki, W., Krejany, E.O., Noviyanti, R., et al., (2002). Transcription of multiple var genes by individual, trophozoite-stage Plasmodium falciparum cells expressing a chondroitin sulphateA binding phenotype. Molecular Microbiology, 43:1285-1293.

Elsasser, S.J., and Arcy, S.D., (2013). Towards a mechanism for histone chaperones. Biochimica et Biophyscica Acta (BBA), 18(9):211-221.

Frank, M., Dzikowski, R., Amulic, B., and Deitsch, K., (2007). Variable switching rates of malaria virulence genes are associated with chromosomal position. Molecular Microbiology, 64: 1486-1498.

Goel, S., Muthusamy, A., Miao, J., Cui, L., Winzeler, E.A., et al., (2014). Targeted disruption of a ring infected erythrocyte surface antigen (RESA)-like export protein gene in Plasmodium falciparum confers stable chrondroitin 4-sulfate cytoadherence capacity. The Journal of Biology Chemistry, M114.615393.

Gupta, A. P., Chin, W.H., Zhu, L., Mok, S., Luah, Y., et al., (2013). Dynamic epigenetic regulation of gene expression during the life cycle of malaria parasite Plasmodium falciparum. PLOS Pathogens, 9(2): e1003170.

Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H., (?). Detection of specific polymerase chain reaction product by utilizing the 5’ to 3’ exonuclease activity of Thermus aquaticus DNA polymerase. Proceeding of the National Academy of Science, USA, 88:7226-7280.

Kraemer, S.M., and Smith, J.D., (2006). The family affair: var gene, PfEMP1 binding and malaria disease. Current Opinion in Microbiology, 9:374-380.

Kritsiriwuthinan, K., Chaotheing, S., Shaw, P.J., Wongsombat, C., Chavalitshewinkoon-Petmitr, P., et al., (2011). Global gene expression profiling of Plasmodium falciparum in response to the anti-malarial drug pyronaridine. Malaria Journal, 10:242-252.

Kulkarni, P., Shiraishi, T., Rajagopalan, K., Kim, R., Mooney, S.M., et al., (2012). Schematic diagram of an immunotherapeutic approach to treating urological malignancies that utilizes immunogenic peptides corresponding to CTAs. Nature Reviews Urology, 9:386-396.

Kyes, S., Christodoulou, Z., Pinches, R., Kriek, N., Horrocks, P., et al., (2007). Plasmodium falciparum var gene expression is developmentally controlled at the level of RNA polymerase II-mediated transcription initiation. Molecular Microbiology, 63(4):1237-1247.

McW Healthcare, (2008). Available at www.mcwhealthcare.com. Accessed date, 29-10-14.

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Mok, S., Imwong, M., Mackinnon, M.J., Sin, J., Ramadoss, R., et al., (2011). Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription. BioMed Central Genomics, 12:391-405.

Noble, R., Christodoulou, Z., Kyes, S., Pinches, R., Newbold, C.I., et al., (2013). The antigenic switching network of Plasmodium falciparum and its implications for the immune-epidemiology of malaria. E Life Science, 2: 1-19.

Pasternak, N.D., and Dzikowski, R., (2009). PfEMP1: An antigen that plays a key role in the pathogenicity and immune evasion of the malaria parasite Plasmodium falciparum. The International Journal of Biochemistry and cell Biology, 41:1463-1466.

Peters, J.M., Fowler, E.V., Krause, D.R., Cheng, Q., and Gatton, M.L., (2007). Differential changes in Plasmodium falciparum var transcription during adaptation to culture. Journal of Infectious Disease, 195:748-755.

Prado, F., and Aguilera, A., (2005). Partial depletion of histone H4 increases homologous recombination-mediated genetic instability. Molecular and Cellular Biology, 25:1526-1536.

Rai, R., Zhu, L., Chen, H., Gupta, A.P., Sze, S.K., et al., (2014). Genome – wide analysis in Plasmodium falciparum reveals early and late phases of RNA polymerase II occupancy during the infectious cycle. Biomed Central Genomics, 15:959-978.

Scherf, A., Riviere, L., and Lopez-Rubio, J.J., (2008). Snapshot: var gene expression in the malaria parasite. Cell, 134:190.

Sellner, L.N., and Turbett, G.R., (1998). Comparism of three RT-PCR methods. Biotechnologies, 25(2):230-234.

Smith, J.D., Gamain, B., Baruch, D.I., and Kyes, S., (2001). Decoding the language of var gene and Plasmodium falciparum sequestration. Trends in Parasitology, 17(11):538-545.

Steiner, L.A., Maksimova, Y., Schulz, V., Wong, C., Raha, D., et al., (2009). Chromatin architecture and transcription factor binding regulate expression of erythrocyte membrane protein genes. Molecular and Cellular Biology, 29(20):5399-5412.

Thomas, A., (2014). Gene expression analysis. On blackboard. Accesses date 22-10-14.

Volz, J.C., Bartfai, R., Petter, M., Langer, C., Josling, G.A., et al., (2012). PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division. Cell Host and Microbe, 11:7-18.

World Health Organization (WHO) (2014). Vox Media, Inc. accessed date 20-11-14.

Zhang, Y., Jiang, N., Chang, Z., Wang, H., Lu, H., et al., (2014). The var3 genes of Plasmodium falciparum 3D7 strain are differentially expressed in infected erythrocytes. Parasite Journal, 21:19-25.