Genome-wide association studies for severe malaria (SM) have identified 30 genetic variants mostly located in non-coding regions. Here, we aimed to identify potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium (LD) with the malaria-associated genetic variants. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing five ATP2B4 SNPs in LD with rs10900585. We found significant associations between SM and rs10900585 and our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we demonstrated that both individual SNPs and the combination of SNPs had regulatory effects. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Our data demonstrate that severe malaria-associated genetic variants alter the expression of ATP2B4 encoding a plasma membrane calcium-transporting ATPase 4 (PMCA4) expressed on red blood cells. Altering the activity of this regulatory element affects the risk of SM, likely through calcium concentration effect on parasitaemia.

BackgroundHost factors, including host genetic variation, have been shown to influence the outcome of Plasmodium falciparum infection. Genome-wide linkage studies have mapped mild malaria resistance genes on chromosome 6p21, whereas NCR3-412 polymorphism (rs2736191) lying within this region was found to be associated with mild malaria.MethodsBlood samples were taken from 188 Plasmodium falciparum malaria patients (76 mild malaria patients, 85 cerebral malaria patients, and 27 severe non-cerebral malaria patients). NCR3-412 (rs2736191) was analysed by sequencing, and haematological parameters were measured. Finally, their association with clinical phenotypes was assessed.ResultsWe evidenced an association of thrombocytopenia with both cerebral malaria and severe non-cerebral malaria, and of an association of high leukocyte count with cerebral malaria. Additionally, we found no association of NCR3-412 with either cerebral malaria, severe non-cerebral malaria, or severe malaria after grouping cerebral malaria and severe non-cerebral malaria patients.ConclusionsOur results suggest that NCR3 genetic variation has no effect, or only a small effect on the occurrence of severe malaria, although it has been strongly associated with mild malaria. We discuss the biological meaning of these results. Besides, we confirmed the association of thrombocytopenia and high leukocyte count with severe malaria phenotypes.

Genome-wide association studies (GWAS) for severe malaria have identified 30 genetic variants that are mostly located in non-coding regions, with only a few associations replicated in independent populations. In this study, we aimed at identifying potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium with the tagSNPs associated with severe malaria in several populations. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing 5 ATP2B4 SNPs in linkage disequilibrium with the tagSNP rs10900585. We confirmed the association of rs10900585 and also found significant associations of severe malaria with our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we showed that this region had both promoter and enhancer activities and that both individual SNPs and the combination of SNPs had regulatory effects using luciferase reporter assays. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Taken together, our data show that severe malaria-associated genetic variants alter the activity of a promoter with enhancer function. We showed that this regulatory element controls the expression of ATP2B4 that encodes a plasma membrane calcium-transporting ATPase 4 (PMCA4), which is the major calcium pump on red blood cells. Altering the activity of this regulatory element affects the risk of severe malaria probably through calcium concentration effect on parasitaemia.

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Background Genome-wide association studies have identified ATP2B4 as a severe malaria resistance gene. Recently, 8 potential causal regulatory variants have been shown to be associated with severe malaria. Methods Genotyping of rs10900585, rs11240734, rs1541252, rs1541253, rs1541254, rs1541255, rs10751450, rs10751451 and rs10751452 was performed in 154 unrelated individuals (79 controls and 75 mild malaria patients). rs10751450, rs10751451 and rs10751452 were genotyped by Taqman assays, whereas the fragment of the ATP2B4 gene containing the remaining SNPs was sequenced. Logistic regression analysis was used to assess the association between the SNPs and mild malaria. Results The results showed that mild malaria was associated with rs10900585, rs11240734, rs1541252, rs1541253, rs1541254, rs1541255, rs10751450, rs10751451 and rs10751452. The homozygous genotypes for the major alleles were associated with an increased risk of mild malaria. Furthermore, the haplotype containing the major alleles and that containing the minor alleles were the most frequent haplotypes. Individuals with the major haplotypes had a significantly higher risk of mild malaria compared to the carriers of the minor allele haplotype. Conclusions ATP2B4 polymorphisms that have been associated with severe malaria are also associated with mild malaria.

With the growing number of cloud providers offering a variety of on-demand services, the process of adopting cloud and selecting a cloud provider is becoming laborious and time consuming involving complex and lengthy negotiations to mediate user requirements with that of cloud Service Level Agreement (SLA) provision. To make the method more efficient, there is a need for an effective, dynamic, and flexible automated approach on negotiation for resolving conflicts and mediating user requirements in SLAs. We contribute to a novel method for automated negotiation, which draws inspiration from two economic concepts -Pareto optimality and Bayesian updating. We describe a model which implements bilateral and multiple attribute negotiations. We exemplify the effectiveness of the approach using scenarios.

This research paper aims to explore the cultural, political, and social realities of Pakistan in Uzma Aslam Khan’s novel Trespassing. The ethnic conflicts which engulfed Karachi in the 1990s in the aftermath of the Afghan War and Gulf War are also depicted. Mixed approach has been used as a tool in order to highlight different aspects of real life presented in the novel. These approaches are critical discourse analysis, punctuation as prosody of language, code mixing, and color semiotics. Code mixing in the novel gives a glaring picture of Pakistani culture and traditional values at the major level by the use of Urduised words. The application of critical discourse analysis in Trespassing reveals the political unrest, corruption, fantasy for America, individualism and ethnic conflicts which swamped the whole country. The prosody of punctuation illustrates the hidden meanings of the sentences more explicit thus making the societal issues more protuberant. The prosody is concerned with the semantic meaning rather than rhetorical meaning. Color semiotics of both eastern and western culture also objectified and presented a more comprehensive vision of the culture, ideas, and feelings in the novel. Trespassing is a book which is realistic and in all sense an essence of Pakistan and its people.