Long non-coding RNAs are essential for Schistosoma mansoni pairing-dependent adult worm homeostasis and fertility.

The trematode parasite Schistosoma mansoni causes schistosomiasis, which affects over 200 million people worldwide. Schistosomes are dioecious, with egg laying depending on the females' obligatory pairing with males. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential that have been involved in other species with reproduction, stem cell maintenance, and drug resistance. In S. mansoni, we recently showed that the knockdown of one lncRNA affects the pairing status of these parasites. Here, we re-analyzed public RNA-Seq data from paired and unpaired adult male and female worms and their gonads, obtained from mixed-sex or single-sex cercariae infections, and found thousands of differentially expressed pairing-dependent lncRNAs among the 23 biological samples that were compared. The expression levels of selected lncRNAs were validated by RT-qPCR using an in vitro unpairing model. In addition, the in vitro silencing of three selected lncRNAs showed that knockdown of these pairing-dependent lncRNAs reduced cell proliferation in adult worms and their gonads, and are essential for female vitellaria maintenance, reproduction, and/or egg development. Remarkably, in vivo silencing of each of the three selected lncRNAs significantly reduced worm burden in infected mice by 26 to 35%. Whole mount in situ hybridization experiments showed that these pairing-dependent lncRNAs are expressed in reproductive tissues. These results show that lncRNAs are key components intervening in S. mansoni adult worm homeostasis, which affects pairing status and survival in the mammalian host, thus presenting great potential as new therapeutic target candidates.

Long non-coding RNAs as possible therapeutic targets in protozoa, and in Schistosoma and other helminths.

Long non-coding RNAs (lncRNAs) emerged in the past 20 years due to massive amounts of scientific data regarding transcriptomic analyses. They have been implicated in a plethora of cellular processes in higher eukaryotes. However, little is known about lncRNA possible involvement in parasitic diseases, with most studies only detecting their presence in parasites of human medical importance. Here, we review the progress on lncRNA studies and their functions in protozoans and helminths. In addition, we show an example of knockdown of one lncRNA in Schistosoma mansoni, SmLINC156349, which led to in vitro parasite adhesion, motility, and pairing impairment, with a 20% decrease in parasite viability and 33% reduction in female oviposition. Other observed phenotypes were a decrease in the proliferation rate of both male and female worms and their gonads, and reduced female lipid and vitelline droplets that are markers for well-developed vitellaria. Impairment of female worms' vitellaria in SmLINC156349-silenced worms led to egg development deficiency. All those results demonstrate the great potential of the tools and methods to characterize lncRNAs as potential new therapeutic targets. Further, we discuss the challenges and limitations of current methods for studying lncRNAs in parasites and possible solutions to overcome them, and we highlight the future directions of this exciting field.

Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.

Novel Alkyl(aryl)-Substituted 2,2-Difluoro-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides: Synthesis, Antimicrobial Activity, and CT-DNA Binding Evaluations.

The synthesis, antimicrobial activity evaluations, biomolecule-binding properties (DNA), and absorption and emission properties of a new series of (Z)-1,1,1-trichloro-4-alkyl(aryl)amino-4-arylbut-3-en-2-ones (4, 5) and 2,2-difluoro-3-alkyl(aryl)amino-4-aryl-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides (6, 7) in which 3(4)-alkyl(aryl) = H, Me, iso-propyl, n-butyl, C6H5, 4-CH3C6H4, 4-CH3OC6H4, 4-NO2C6H4, 4-FC6H4, 4-BrC6H4, 2-naphthyl, is reported. A series of ß-enaminoketones (4, 5) is synthesized from the O,N-exchange reaction of some amines (3) with (Z)-1,1,1-trichloro-4-methoxy-4-aryl-but-3-en-2-ones (1, 2) at 61-90% yields. Subsequently, reactions of the resulting ß-enaminoketones with an appropriate source of boron (BF3.OEt2) gave the corresponding oxazaborinine derivatives (6, 7) at 50-91% yields. UV-Vis and emission properties of biomolecule-binding properties for the DNA of these new BF2-ß-enamino containing CCl3 units were also evaluated. Some compounds from the present series also exhibited potent antimicrobial effects on various pathogenic microorganisms at concentrations below those that showed cytotoxic effects. Compounds 4d, 4e, 6e, and 6f showed the best results and are very significant against P. zopfii, which causes diseases in humans and animals.

A phytochemical study of the Cuphea glutinosa from Southern Brazil: Na+,K+-ATPase activity inhibition and antioxidant properties.

This study investigated the antioxidant activity of Cuphea glutinosa (CG) and its effect on Na+, K+-ATPase from cardiac muscle. The ethanolic extract showed higher antioxidant capacity compared to aqueous and ethyl acetate fraction. Ethyl acetate fraction showed ß-sitosterol-3-O-ß-glucoside, kaempferol, quercetin, isoquercetin, gallic acid methyl ester, and gallic acid. The ethanolic extract also reduced the Na+,K+-ATPase activity. CG presented a promising antioxidant activity and inhibitory effect on the Na+, K+-ATPase activity, supporting biochemical evidences the popular use of this plant in the treatment of heart failure.

Synthesis, antimicrobial activity, and QSAR studies of furan-3-carboxamides.

The synthesis and characterization of a new series of furan-3-carboxamides, from the aromatization of 4-trichloroacetyl-2,3-dihydrofuran to 3-trichloroacetyl furan followed by nucleophilic displacement of the trichloromethyl group or the corresponding carboxylic acid chloride by nitrogen-containing compounds, is presented. Preliminary in vitro antimicrobial activity of the title compounds was assessed against a panel of microorganisms including yeast, filamentous fungi, bacteria, and alga. Some of the furan-3-carboxamides exhibited significant in vitro antimicrobial activity. QSAR investigation was applied to find a correlation between the different physicochemical parameters of the compounds studied and their biological activity.

Synthesis and antimicrobial activity of new (4,4,4-trihalo-3-oxo-but-1-enyl)-carbamic acid ethyl esters, (4,4,4-trihalo-3-hydroxy-butyl)-carbamic acid ethyl esters, and 2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters.

This work presents a three-step synthesis of a new series of 4-substituted 2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters, from beta-alkoxyvinyl trihalomethyl ketones of general formula X3C-C(O)-CH=C(R)-OR(1), where R = H, Me, Ph, and 4-Me-Ph; R(1) = Me and Et; and X = F and Cl. The Michael addition-substitution of the ethyl carbamate on beta-alkoxyvinyl trihalomethyl ketones furnished the corresponding (4,4,4-trihalo-3-oxo-but-1-enyl)-carbamic acid ethyl esters. These compounds underwent reduction with NaBH4 leading to the respective (4,4,4-trihalo-3-hydroxy-butyl)-carbamic acid ethyl esters. The 3-hydroxy-butyl carbamates were submitted to cyclization reaction with triphosgene to give a series of 4-substituted 2-oxo-6-trihalomethyl-[1,3]oxazinane-3-carboxylic acid ethyl esters. The in vitro antimicrobial activity, of some of the three new series of the title compounds, was assessed against a panel of microorganisms including yeast like fungi, bacteria, and algae, and their minimal inhibitory concentration and minimal fungicidal, bactericidal, and algacidal concentrations were determined. Some of the analyzed carbamates exhibited significant in vitro antimicrobial activity.