BackgroundSchistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.Methodology and ResultsWe have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.ConclusionsAuthentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.

Many microbial pathogens, including the malaria parasite Plasmodium falciparum, vary surface protein expression to evade host immune responses. P. falciparium antigenic variation is linked to var gene family-encoded clonally variant surface protein expression. Mututally exclusive var gene expression is partially controlled by spatial positioning; silent genes are retained at distinct perinuclear sites and relocated to transcriptionally active locations for monoallelic expression. We show that var introns can control this process and that var intron addition relocalizes episomes from a random to a perinuclear position. This var intron-regulated nuclear tethering and repositioning is linked to an 18 bp nuclear protein-binding element that recruits an actin protein complex. Pharmacologically induced F-actin formation, which is restricted to the nuclear periphery, repositions intron-carrying episomes and var genes and disrupts mutually exclusive var gene expression. Thus, actin polymerization relocates var genes from a repressive to an active perinuclear compartment, which is crucial for P. falciparium phenotypic variation and pathogenesis.

The artemisinin (ART), discovered in China, has been widely used against malaria in China over the last 30 years. Understanding the emergence and origin of ART resistance in China is therefore of great interest. We analyzed 111 culture-adapted isolates of P. falciparum from China-Myanmar (CM) border for their susceptibility to dihydroartemisinin using the ring stage survival assay (RSA0−3h) and genotyped their K13 genes. Of the isolates, 59 had a wild type of the K13 marker and a median ring survival rate of 0.26% (P95 = 1.005%). Among the remaining isolates harboring single mutations in the K13 marker, 26 survived at >P95(median survival rate = 2.95%). Further, we genotyped the K13 gene of 693 isolates collected from different regions in China and China-Myanmar/Thai-Cambodia/Thai-Myanmar (CM/TC/TM) borders, 308 (44.4%) had K13 mutations and marked differences in the patterns of K13 mutations were observed between the CM and the TC/TM borders. A network diagram showed that majority of the K13 mutant alleles from the CM border clustered together including those harboring the high resistant-associated R539T mutations. The resistant parasites carrying distinct halplotypes suggested the multiple indigenous origins of the resistant alleles, which highlight the importance of surveillance of resistance in all malaria endemic areas where ART has been introduced.

Background: The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs) are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi) and posttranscriptional gene silencing (PTGS) in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs.

BackgroundMutations in the Plasmodium falciparum k13 gene are associated with artemisinin (ART) resistance. However, it is unclear whether the F446I mutation, the most prevalent allele at the China–Myanmar border and north of Myanmar, is associated with ART resistance. Therefore, the aim of this study was to investigate the role of this mutation in ART resistance by generating transgenic parasites expressing the F446I mutant allele.MethodsThe transgenic parasites carrying the F446I or C580Y mutation in both 3D7 and FCC1/HN isolates were generated by single crossing-over recombination and verified using PCR and gene sequencing. The ring-stage survival assay of 0–3 h (RSA0–3 h) was used to evaluate ART susceptibility of the transgenic parasites in vitro.ResultsFour transgenic parasite lines named 3D7F446I mut, 3D7C580Y mut, FCC1/HNF446I mut and FCC1/HNC580Y mut were successfully generated. These parasite lines showed no changes in the expression level of k13 when compared with their parent parasite isolates. However, introduction of the F446I mutation in k13 of the 3D7 and FCC1/HN isolates led to elevated ring survival rates detected using RSA0–3 h when subjected to both 700 and 20 nM concentrations of dihydroartemisinin. The survival rates were similar to those detected in the parasite lines with the C580Y mutation.ConclusionsInsertion of the F446I mutation in k13 led to increased ring survival, suggesting that this mutation may be associated with ART resistance and could be used as a molecular marker for monitoring ART-resistant parasites. The results also highlights the importance of surveillance of F446I mutants for containing the resistant parasite.