No abstract

The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China's Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO's Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by ‘border malaria’ and ‘forest malaria’ with high transmission occurring along international borders and in forests or forest fringes, respectively. ‘Border malaria’ is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and P. vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination.

cArtemisinin resistance in Plasmodium falciparum parasites in Southeast Asia is a major concern for malaria control. Its emergence at the China-Myanmar border, where there have been more than 3 decades of artemisinin use, has yet to be investigated. Here, we comprehensively evaluated the potential emergence of artemisinin resistance and antimalarial drug resistance status in P. falciparum using data and parasites from three previous efficacy studies in this region. These efficacy studies of dihydroartemisinin-piperaquine combination and artesunate monotherapy of uncomplicated falciparum malaria in 248 P. falciparum patients showed an overall 28-day adequate clinical and parasitological response of >95% and day 3 parasite-positive rates of 6.3 to 23.1%. Comparison of the 57 K13 sequences (24 and 33 from day 3 parasite-positive and -negative cases, respectively) identified nine point mutations in 38 (66.7%) samples, of which F446I (49.1%) and an N-terminal NN insertion (86.0%) were predominant. K13 propeller mutations collectively, the F446I mutation alone, and the NN insertion all were significantly associated with day 3 parasite positivity. Increased ring-stage survival determined using the ring-stage survival assay (RSA) was highly associated with the K13 mutant genotype. Day 3 parasite-positive isolates had ϳ10 times higher ring survival rates than day 3 parasitenegative isolates. Divergent K13 mutations suggested independent evolution of artemisinin resistance. Taken together, this study confirmed multidrug resistance and emergence of artemisinin resistance in P. falciparum at the China-Myanmar border. RSA and K13 mutations are useful phenotypic and molecular markers for monitoring artemisinin resistance.

Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis.colorectal cancer R eversible tyrosine phosphorylation, which is coordinately controlled by protein tyrosine kinases (PTKs) and phosphatases (PTPs), governs numerous signaling pathways that regulate cell proliferation, apoptosis, adhesion, and migration. Over the last two decades, many PTKs have been found to be mutated in a variety of different tumor types (reviewed in ref. 1). In contrast to PTKs, the role of PTPs in tumorigenesis is underexplored. To systematically evaluate possible roles of PTPs in tumorigenesis, we used a high throughput molecular and bioinformatics approach to detect genetic alterations of the tyrosine phosphatase gene family in colorectal cancers (CRCs) (2). Among the six mutated PTPs that we identified, protein tyrosine phosphatase receptor-type T (PTPRT), also known as PTPρ, was the most frequently mutated (2). In addition, we and others found that PTPRT is also mutated in lung, stomach, and skin cancers (2, 3). The spectrum of mutations, which includes nonsense mutations and frameshifts, suggested that these mutations were inactivating (2). Biochemical analyses demonstrated that missense mutations in the catalytic domains of PTPRT diminished its phosphatase activity, whereas overexpression of PTPRT inhibited CRC cell growth (2). Taken together, these studies suggest that PTPRT normally acts as a tumor suppressor gene. In light of these data, it is important to identify the functionally significant substrates of PTPRT as well as to elucidate the signal transduction pathways regulated by this phosphatase. Here, we report that paxillin, an adaptor protein involved in cell adhesion, migration, proliferation, and apoptosis (4, 5), is a direct substrate of PTPRT and that phospho-paxillin has oncogenic properties. We further demonstrate that in an in vivo model, PTPRT is both a potent tumor suppressor gene and a key regulator of colonic ph...

BackgroundThe recent emergence and spread of artemisinin resistance in the Greater Mekong Subregion poses a great threat to malaria control and elimination. A K13-propeller gene (K13), PF3D7_1343700, has been associated lately with artemisinin resistance both in vitro and in vivo. This study aimed to investigate the K13 polymorphisms in Plasmodium falciparum parasites from the China-Myanmar border area where artemisinin use has the longest history.MethodsA total of 180 archived P. falciparum isolates containing 191 parasite clones, mainly collected in 2007–2012 from the China-Myanmar area, were used to obtain the full-length K13 gene sequences.ResultsSeventeen point mutations were identified in 46.1% (88/191) parasite clones, of which seven were new. The F446I mutation predominated in 27.2% of the parasite clones. The C580Y mutation that is correlated with artemisinin resistance was detected at a low frequency of 1.6%. Collectively, 43.1% of the parasite clones contained point mutations in the kelch domain of the K13 gene. Moreover, there was a trend of increase in the frequency of parasites carrying kelch domain mutations through the years of sample collection. In addition, a microsatellite variation in the N-terminus of the K13 protein was found to have reached a high frequency (69.1%).ConclusionsThis study documented the presence of mutations in the K13 gene in parasite populations from the China-Myanmar border. Mutations present in the kelch domain have become prevalent (>40%). A predominant mutation F446I and a prevalent microsatellite variation in the N-terminus were identified, but their importance in artemisinin resistance remains to be elucidated.

Quinine resistance (QNR) in Plasmodium falciparum has been detected in many regions of the world where malaria is endemic. Genetic polymorphisms in at least four genes are implicated in QN susceptibility, and their significance often depends on the genetic background of the parasites. In this study, we have culture-adapted 60 P. falciparum clinical isolates from the China-Myanmar border and assessed their in vitro responses to QN. Our results showed that >50% of the parasite isolates displayed reduced sensitivity to QN, with a half-maximal inhibitory concentration (IC 50 ) above 500 nM. Genotyping of pfcrt found that an overwhelming proportion of the parasite population had the chloroquine-resistant genotype, whereas pfmdr1 mutation genotypes and gene amplification were rare. Genotyping of the P. falciparum Na ؉ /H ؉ exchanger gene (pfnhe1) at the minisatellite ms4760 locus identified 10 haplotypes. Haplotype 7, which harbors three copies of the DNNND repeat, was the most predominant, accounting for nearly half of the parasite isolates. Correlation studies did not reveal significant associations of the polymorphisms in pfcrt and pfmdr1 genes with QN response. However, the ms4760 haplotypes were highly associated with in vitro QN responses. In particular, parasite isolates with an increased DNNND copy number tended to have significantly reduced QN susceptibility, whereas parasite isolates with a higher NHNDNHNNDDD copy number had increased QN susceptibility. This study provided further support for the importance of pfnhe1 polymorphisms in influencing QNR in P. falciparum.

The recent resurgence of Plasmodium vivax malaria requires close epidemiological surveillance and monitoring of the circulating parasite populations. In this study, we developed a combination of polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) method to investigate the genetic diversity of the P. vivax merozoite surface protein 3β (PvMSP3β) gene among four Asian parasite populations representing both tropical and temperate strains with dramatic divergent relapse patterns (N = 143). Using P. vivax field isolates from symptomatic patients, we have validated the feasibility of this protocol in distinguishing parasite genotypes. We have shown that PCR alone could detect three major size polymorphisms of the PvMSP3β gene, and restriction analysis detected a total of 12 alleles within these Asian samples. Samples from different geographical areas differed dramatically in their PvMSP3β allele composition and frequency, indicating that complex, yet different parasite genotypes were circulating in different endemic areas. This protocol allowed easy detections of multiple infections, which reached 20.5% in the samples from Thailand. It is interesting to note that samples from one temperate site in China collected during a recent outbreak of the disease also showed a high level of genetic diversity with multiple infections accounting for 5.6% of the samples. When combined with the PvMSP3α locus, this method provides better capability in distinguishing P. vivax genotypes and detecting mixed genotype infections.

Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr. Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine – an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy.