Transfer RNA (tRNA)-derived fragments (tRFs) have been widely identified in nature, functioning in diverse biological and pathological situations. Yet, the presence of these small RNAs in Plasmodium spp. remains unknown. Systematic identification and characterization of tRFs is therefore highly needed to understand further their roles in Plasmodium parasites, particularly in the virulent Plasmodium falciparum parasite. Genome-wide small RNAs with sizes ranging from 18–30 nucleotides from P. falciparum were deep-sequenced via Illumina HiSeq 2000 technology. In-depth analysis revealed the presence of a vast number of small RNAs originating from tRNA-coding genes, responsible for 22.4% of the total reads as the second predominant group. Three P. falciparum-derived tRF types (ptRFs) were identified as 5'ptRFs, mid-ptRFs and 3'ptRFs. The majority (90%) of ptRFs were derived from tRNAs that coded eight amino acids: Pro, Phe, Asn, Gly, Cys, Gln, His and Ala. Stem-loop reverse transcription polymerase chain reaction further confirmed the presence of tRFs in the blood stages of P. falciparum. Four new motifs with an enriched G/C feature were determined at cleavage sites that might guide the generation of ptRFs. To our knowledge, this is the first report of a genome-wide investigation of ptRFs from Plasmodium species. The identification of ptRFs reveals a complex small RNA system manipulated by the malaria parasite, and might promote research on the function of tRFs in the pathogenesis of Plasmodium infections.

Background: Small non-coding RNAs play critical regulatory roles in post-transcription. However, their characteristics in Trichomonas vaginalis, the causative agent of human sexually transmitted trichomoniasis, still remain to be determined. Methods: Small RNA transcriptomes from Trichomonas trophozoites were deep sequenced using the Illumina NextSeq 500 system and comprehensively analyzed to identify Trichomonas microRNAs (miRNAs) and transfer RNA (tRNA)-derived small RNAs (tsRNAs). The tsRNA candidates were confirmed by stem-loop quantitative reverse transcription-PCR, and motifs to guide the cleavage of tsRNAs were predicted using the GLAM2 algorithm. Results: The miRNAs were found to be present in T. vaginalis but at an extremely low abundance (0.0046%). Three categories of endogenous Trichomonas tsRNAs were identified, namely 5′tritsRNAs, mid-tritsRNAs and 3′tritsRNAs, with the 5′tritsRNAs constituting the dominant category (67.63%) of tsRNAs. Interestingly, the cleavage site analysis verified both conventional classes of tRNA-derived fragments (tRFs) and tRNA-halves in tritsRNAs, indicating the expression of tRNA-halves in the non-stress condition. A total of 25 tritsRNAs were experimentally confirmed, accounting for 78.1% of all tested candidates. Three motifs were predicted to guide the production of tritsRNAs. The results prove the expression of tRFs and tRNA-halves in the T. vaginalis transcriptome. Conclusions: This is the first report of genome-wide investigation of small RNAs, particularly tsRNAs and miRNAs, from Trichomonas parasites. Our findings demonstrate the expression profile of tsRNAs in T. vaginalis, while miRNA was barely detected. These results may promote further research aimed at gaining a better understanding of the evolution of small non-coding RNA in T. vaginalis and their functions in the pathogenesis of trichomoniasis. Graphical

Malaria chemotherapy is greatly threatened by the recent emergence and spread of resistance in the Plasmodium falciparum parasite against artemisinins and their partner drugs. Therefore, it is an urgent priority to develop new antimalarials. Plasmepsin V (PMV) is regarded as a superior drug target for its essential role in protein export. In this study, we performed virtual screening based on homology modeling of PMV structure, molecular docking and pharmacophore model analysis against a library with 1,535,478 compounds, which yielded 233 hits. Their antimalarial activities were assessed amongst four non-peptidomimetic compounds that demonstrated the promising inhibition of parasite growth, with mean IC₅₀ values of 6.67 μM, 5.10 μM, 12.55 μM and 8.31 μM. No significant affection to the viability of L929 cells was detected in these candidates. These four compounds displayed strong binding activities with the PfPMV model through H-bond, hydrophobic, halogen bond or π-π interactions in molecular docking, with binding scores under −9.0 kcal/mol. The experimental validation of molecule-protein interaction identified the binding of four compounds with multiple plasmepsins; however, only compound 47 showed interaction with plasmepsin V, which exhibited the potential to be developed as an active PfPMV inhibitor.

Plasmodium vivax remains the predominant species at the China–Myanmar border, imposing a major challenge to the recent gains in regional malaria elimination. To closely supervise the emerging of drug resistance in this area, we surveyed the variations in genes potentially correlated with drug resistance in P. vivax parasite and the possible drug selection with time. A total of 235 P. vivax samples were collected from patients suffering uncomplicated malaria at Yingjiang, Tengchong, and Longling counties, and Nabang port in China, Yunnan province, and Laiza sub-township in Myanmar, from 2008 to 2017. Five potential drug resistance genes were amplified utilizing nested-PCR and analyzed, including pvdhfr, pvdhps, pvmdr1, pvcrt-o, and pvk12. The Pearson’s Chi-squared test or Fisher’s exact test were applied to determine the statistical frequency differences of mutations between categorical data. The pvdhfr F57I/L, S58R, T61M and S117T/N presented in 40.6%, 56.7%, 40.1%, and 56.0% of the sequenced P. vivax isolates, and these mutations significantly decreased with years. The haplotype formed by these quadruple mutations predominated in Yingjiang, Tengchong, Longling and Nabang. While a mutation H99S/R (56.6%) dominated in Laiza and increased with time. In pvdhps, the A383G prevailed in 69.2% of the samples, which remained the most prevalent haplotype. However, a significant decrease of its occurrence was also noticed over the time. The S382A/C and A553G existed in 8.4% and 30.8% of the isolates, respectively. In pvmdr1, the mutation Y976F occurred at a low frequency in 5/232 (2.2%), while T958M was fixed and F1076L was approaching fixed (72.4%). The K10 insertion was detected at an occurrence of 33.2% in pvcrt-o, whereas there was no significant difference among the sites or over the time. No mutation was identified in pvk12. Mutations related with resistance to antifolate drugs are prevalent in this area, while their frequencies decrease significantly with time, suggestive of increased susceptibility of P. vivax parasite to antifolate drugs. Resistance to chloroquine (CQ) is possibly emerging. However, since the molecular mechanisms underneath CQ resistance is yet to be better understood, close supervision of clinical drug efficiency and continuous function investigation is urgently needed to alarm drug resistance.

Background: The emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs. Methods: Modified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs). Results: A modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 10⁴ parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 10⁴ parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 10⁴ parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003). Conclusions: This study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination. Graphical

Background: Plasmodium vivax remains the predominant species at the China–Myanmar border, imposing a major challenge to the recent gains in regional malaria elimination. To closely supervise the emerging of drug resistance in this area, we surveyed the variations in genes potentially correlated with drug resistance in P. vivax parasite and the possible drug selection with time.Methods: A total of 235 P. vivax samples were collected from patients suffering uncomplicated malaria at Yingjiang, Tengchong, and Longling counties, and Nabang port in China, Yunnan province, and Laiza township in Myanmar, during 2008 to 2017. Five potential drug resistance genes were amplified utilizing nested-PCR and analyzed, including pvdhfr, pvdhps, pvmdr1, pvcrt-o, and pvk12. Results: The pvdhfr F57I/L, S58R, T61M and S117T/N presented in 40.64%, 56.68%, 40.11%, and 56.04% of the sequenced P. vivax isolates, and these mutations significantly decreased with years. The haplotype formed by these quadruple mutations predominated at Yingjiang, Tengchong, Longling and Nabang. While a mutation H99S/R (56.59%) dominated at Laiza and increased with time. In pvdhps, the A383G prevailed in 69.16% of the samples, which remained the most prevalent haplotype. However, a significant decrease of its occurrence was also noticed over the time. The S382A/C and A553G existed in 8.37% and 30.84% of the isolates, respectively. In pvmdr1, the mutation Y976F occurred at a low frequency in 5/232 (2.16%), while T958M was fixed and F1076L was approaching fixed (72.41%). The K10 insertion was detected at an occurrence of 33.17% in pvcrt-o, whereas there was no significant difference among the sites or over the time. No mutation was identified in pvk12.Conclusions: Mutations related with resistance to antifolate drugs are prevalent in this area as expected, while their frequencies decrease significantly with time, suggestive of increased susceptibility of P. vivax parasite to antifolate drugs. Resistance to CQ is possibly emerging. However, since the molecular mechanisms underneath CQ resistance is yet to be better understood, close supervision of clinical drug efficiency and continuous function investigation is urgently needed to alarm drug resistance.