Detection of Developmental Asexual Stage-Specific RNA Editing Events in Plasmodium falciparum 3D7 Malaria Parasite.

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control.

Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System.

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA.

Antiplasmodial and interferon-gamma-modulating activities of the aqueous extract of stone breaker (Phyllanthus niruri Linn.) in malaria infection.

Plasmodium falciparum parasites are the primary cause of malaria across Africa. The problem of drug resistance to malaria is ever growing and novel therapeutic strategies need to be developed, particularly those targeting the parasite and also the host or host-pathogen interaction. Previous studies have shown that the development of cerebral malaria (CM) is related to dysregulation of the immune system in a murine malaria model of experimental cerebral malaria. It involves a complex interaction of events and interferon-gamma seems to be the unifying factor. Therefore, the antiplasmodial activity targeting the parasite and immunomodulatory strategies that reduce overall host inflammation, with IFN-γ in focus, could delay CM onset and prove beneficial in malaria infection therapy. Phyllanthus niruri is used to treat fever and other symptoms of malaria in Nigeria. Its modes of action as an anti-malarial remedy have not been exhaustively investigated. This study therefore examined the aqueous extract of P. niruri (PE) for its antiplasmodial activity in vitro using the Plasmodium falciparum HB3 strain. Furthermore, in vivo murine malaria model using the Plasmodium berghei ANKA strain was used to investigate its anti-malarial effects. We showed that PE has multiple anti-malarial effects, including anti-parasitic and host immunomodulatory activities. Co-culture of P. falciparum with PE and some of its phytoconstituents drastically reduced parasite number. PE also decreased parasitemia, and increased the survival of infected mice. We also observed that the integrity of the blood-brain barrier was maintained in the PE-treated mice. The results confirmed that PE showed moderate antiplasmodial activity. In vivo murine malaria model using P. berghei ANKA for experimental cerebral malaria revealed that PE suppressed parasite growth, and modulate the production of interferon-gamma. The findings demonstrate that PE affects malaria progression, targeting parasites and host cells.

Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase.

Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5' than for the 3'A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

Theileria annulata seroprevalence among different cattle breeds in Rajshahi Division, Bangladesh.

An epidemiological survey of Theileria annulata infection was undertaken in a cattle population in Rajshahi Division, Bangladesh. The local cattle breeds from the area (North Bengal Gray and Deshi) and crosses between the local breeds and Holstein cattle were predominantly screened. In total, 192 cattle serum samples were collected in two areas of Rajshahi Division, the Rajshahi District (n=147) and Natore District (n=45). The samples were screened with an enzyme-linked immunosorbent assay using T. annulata surface protein (TaSP) as the antigen. The seroprevalence was 80.0% (36/45) in Natore and 20.4% (30/147) in Rajshahi. A logistic regression analysis showed that the sampling location was significantly associated with seropositivity, whereas age, sex and breed were not. Although the logistic regression analysis did not show a linear dependence on age, we considered age-specific seroprevalence separately in the two districts. Seroprevalence did not differ significantly among age categories in the Natore District. In contrast, all the cattle <1 year old in the Rajshahi District were seronegative (11/11). Seroprevalence in the 1- and 2-year-old cattle was significantly lower in the Rajshahi District than in the Natore District. In the older age categories (3, 4 and >5 years), seroprevalence did not differ significantly between the Natore and Rajshahi Districts. These results suggest that the cattle in the Rajshahi District were sporadically exposed to T. annulata, whereas most cattle in the Natore District became infected during an early phase of life.

Age-specificity of Toxoplasma gondii seroprevalence in sheep, goats and cattle on subsistence farms in Bangladesh.

Toxoplasma gondii is a zoonotic protozoan parasite that infects humans and domestic animals. In this study, the seroprevalence of T. gondii antibodies was investigated using serum samples collected from 83 sheep, 146 goats and 37 cattle from a dozen subsistence farms in Bangladesh. Fifty-eight out of 83 sheep (69.9%), 89 out of 146 goats (61.0%) and 10 out of 37 cattle (27.0%) were seropositive for the parasite. Seroprevalence in young goats (<1 year old) was significantly lower than that of the adult goats (>1 year old). In contrast, seroprevalence for young and adult sheep was similar. These results indicate that acquired infection with T. gondii occurs in this region of Bangladesh, at least among goats.