Stable expression of red fluorescent protein-blasticidin deaminase fusion gene (rfp-bsd) as a selectable marker for DNA transfection in Babesia ovata.

The red fluorescent protein(rfp)-blasticidin deaminase(bsd) fusion gene was transfected into Babesia ovata by electroporation with the plasmid DNA and selected with 15 µg/mL of blasticidin S under the in vitro culture condition. The transfected parasite with episomal DNA was selected and cultured for further analysis based on the presence of the rfp-bsd fusion gene by PCR and expression of the fusion protein by immunofluorescence antibody test under fluorescence microscopy for 2 months after the transfection. The results are the first, to our knowledge, to demonstrate the expression and stability of the episomal rfp-bsd fusion gene under the control of actin promoter as a selectable marker for the transfection system in B. ovata.

Evaluation of the antiplasmodial efficacy of synthetic 2,5-diphenyloxazole analogs of compounds naturally derived from Oxytropis lanata.

The persistent prevalence and dissemination of drug-resistant malaria parasites continue to challenge the progress of malaria eradication efforts. As a result, there is an urgent need to search for and develop innovative therapies. In this study, we screened synthetic 2,5-diphenyloxazole analogs from Oxytropis lanata. Among 48 compounds, 14 potently inhibited the proliferation of P. falciparum strains 3D7 (chloroquine-sensitive) and K1 (multidrug-resistant) in vitro, exhibited IC50 values from 3.38 to 12.65 µM and 1.27-6.19 µM, respectively, and were toxic to human foreskin fibroblasts at 39.53-336.35 µM. Notably, Compounds 31 (2-(2',3'-dimethoxyphenyl)-5-(2″-hydroxyphenyl)oxazole) and 32 (2-(2',3'-dimethoxyphenyl)-5-(2″-benzyloxyphenyl)oxazole) exhibited the highest selectivity indices (SIs) against both P. falciparum strains (3D7/K1), with values > 40.20/>126.58 and > 41.27/> 59.06, respectively. In the IC50 speed and stage-specific assays, Compounds 31 and 32 showed slow action, along with distinct effects on the ring and trophozoite stages. Microscopy observations further revealed that both compounds impact the development and delay the progression of the trophozoite and schizont stages in P. falciparum 3D7, especially at concentrations 100 times their IC50 values. In a 72-h in vitro exposure experiment at their respective IC80 in P. falciparum 3D7, significant alterations in parasitemia levels were observed compared to the untreated group. In Compound 31-treated cultures, parasites shrank and were unable to reinvade red blood cells (RBCs) during an extended 144-h incubation period, even after compound removal from the culture. In vivo assessments were conducted on P. yoelii 17XNL-infected mice treated with Compounds 31 and 32 at 20 mg/kg administered once daily for ten days. The treated groups showed statistically significant lower peaks of parasitemia (Compound 31-treated: trial 1 12.7%, trial 2 15.8%; Compound 32-treated: trial 1 12.7%, trial 2 14.0%) compared to the untreated group (trial 1 21.7%, trial 2 28.3%). These results emphasize the potential of further developing 2,5-diphenyloxazoles as promising antimalarial agents.

Retinal ferroptosis as a critical mechanism for the induction of retinochoroiditis during ocular toxoplasmosis.

Toxoplasmosis is a major infectious disease, affecting approximately one-third of the world's population; its main clinical manifestation, ocular toxoplasmosis (OT), is a severe sight-threatening disease. Nevertheless, the diagnosis of OT is based on clinical findings, which needs improvement, even with biochemical tests, such as polymerase chain reaction and antibody detections. Furthermore, the efficacy of OT-targeted treatment is limited; thus, additional measures for diagnosis and treatments are needed. Here, we for the first time report a significantly reduced iron concentration in the vitreous humor (VH) of human patients infected with OT. To obtain further insights into molecular mechanisms, we established a mouse model of T. gondii infection, in which intravitreally injected tracer 57Fe, was accumulated in the neurosensory retina. T. gondii-infected eyes showed increased lipid peroxidation, reduction of glutathione peroxidase-4 expression and mitochondrial deformity in the photoreceptor as cristae loss. These findings strongly suggest the involvement of ferroptotic process in the photoreceptor of OT. In addition, deferiprone, an FDA-approved iron chelator, reduced the iron uptake but also ameliorated toxoplasma-induced retinochoroiditis by reducing retinal inflammation. In conclusion, the iron levels in the VH could serve as diagnostic markers and iron chelators as potential treatments for OT.

Antiplasmodial Activity Evaluation of a Bestatin-Related Aminopeptidase Inhibitor, Phebestin.

The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 µM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.

Effect of α-Tocopheryloxy Acetic Acid on the Infection of Mice with Plasmodium berghei ANKA In Vivo and Humans with P. falciparum In Vitro.

PURPOSE: Malarial parasites are susceptible to oxidative stress. The effects of α-tocopheryloxy acetic acid (α-TEA), a vitamin E analog, on infection by Plasmodium berghei ANKA and P. falciparum in mice and human red blood cells (RBCs), respectively, were examined in this study. METHODS: For in vivo studies in mice, RBCs infected with P. berghei ANKA were inoculated via intraperitoneal injection and α-TEA was administered to C57BL/6 J male mice after infection. The blood-brain barrier (BBB) permeability was examined by Evans blue staining in experimental cerebral malaria at 7 days after infection. The in vitro inhibitory effect of α-TEA on P. falciparum 3D7 (chloroquine-sensitive strain) and K1 (multidrug-resistant strain) was tested using a SYBR Green I-based assay. RESULTS: When 1.5% α-TEA was administered for 14 days after infection, 88% of P. berghei ANKA-infected mice survived during the experimental period. Nevertheless, all the control mice died within 12 days of infection. Furthermore, the Evans blue intensity in α-TEA-treated mice brains was less than that in untreated mice, indicating that α-TEA might inhibit the destruction of the BBB and progression of cerebral malaria. The in vitro experiment revealed that α-TEA inhibited the proliferation of both the 3D7 and K1 strains. CONCLUSION: This study showed that α-TEA is effective against murine and human malaria in vivo and in vitro, respectively. Although α-TEA alone has a sufficient antimalarial effect, future research could focus on the structure-activity relationship to achieve better pharmacokinetics and decrease the cytotoxicity and/or the combined effect of α-TEA with existing drugs. In addition, the prophylactic antimalarial activity of premedication with α-TEA may also be an interesting perspective in the future.

Evaluation of Mongolian compound library for potential antimalarial and anti-Toxoplasma agents.

179 compounds in a Mongolian compound library were investigated for their inhibitory effect on the in vitro growth of Plasmodium falciparum and Toxoplasma gondii. Among these compounds, brachangobinan A at a half-maximal inhibition concentration (IC50) of 2.62 µM and a selectivity index (SI) of 27.91; 2-(2'-hydroxy-5'-O-methylphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (IC50 3.58 µM and SI 24.66); chrysosplenetin (IC50 3.78 µM and SI 15.26); 4,11-di-O-galloylbergenin (IC50 3.87 µM and SI 13.38); and 2-(2',5'-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (IC50 6.94 µM and SI 11.48) were identified as potential inhibitors of P. falciparum multiplication. Additionally, tricin (IC50 12.94 µM and SI > 23.40) was identified as a potential inhibitor of T. gondii multiplication. Our findings represent a good starting point for developing novel antimalarial and anti-Toxoplasma therapeutics from Mongolian compounds.

Colletofurans A-E, 1-Octyl-1,3-dihydroisobenzofuran Derivatives from Colletotrichum boninense AM-12-2.

3-(3-Hydroxy-2-(hydroxymethyl)phenyl)propanoic acid, 2-hydroxymethyl-3-hydroxy-(E)-cinnamic acid, and colletofurans A-E were isolated from Colletotrichum boninense AM-12-2. Colletofurans A-E are the first natural compounds featuring a 1-octyl-1,3-dihydroisobenzofuran core. Their structures were initially established by 1D/2D-NMR and HRESITOFMS. Mosher's ester method was used to determine the absolute configurations of secondary alcohols in colletofurans A-C. The structures of colletofurans A and B-E were further confirmed by DFT GIAO calculations and the X-ray crystalline sponge method, respectively.