Effect of Rodent Control Program on Incidence of Zoonotic Cutaneous Leishmaniasis, Iran.

We report the effect of a rodent control program on the incidence of zoonotic cutaneous leishmaniasis in an endemic region of Iran. A 1-year interruption in rodent control led to 2 years of increased incidence of zoonotic cutaneous leishmaniasis. Restarting rodent control led to a decline of zoonotic cutaneous leishmaniasis.

Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Circadian Regulation of Leishmania Parasite Internalisation in Macrophages and Downstream Cellular Events.

Leishmania spp. parasites use macrophages as a host cell during infection. As a result, macrophages have a dual role: clearing the parasite as well as acting as host cells. Recently, studies have shown that macrophages harbour circadian clocks, which affect many of their functions such as phagocytosis, receptor expression and cytokine release. Interestingly, Leishmania major infection in hosts was also shown to be under circadian control. Therefore, we decided to investigate what underlies the rhythms of L. major infection within macrophages. Using a culture model of infection of bone marrow-derived macrophages with L. major promastigotes, we show that the parasites are internalised into macrophages with a 24-h variation dependent on a functional circadian clock in the cells. This was associated with a variation in the number of parasites per macrophage. The cell surface expression of parasite receptors was not controlled by the cells' circadian clock. In contrast, the expression of the components of the endocytic pathway, EEA1 and LC3b, varied according to the time of infection. This was paralleled by variations in parasite-induced ROS production as well as cytokine tumour necrosis factor α. In summary, we have uncovered a time-dependent regulation of the internalisation of L. major promastigotes in macrophages, controlled by the circadian clock in these cells, as well as subsequent cellular events in the endocytic pathway, intracellular signalling and cytokine production.

Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites.

RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB, and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first published draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131, and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major. KEY POINTS: • A four-subunit TFIIIC complex is present in T. brucei and L. major • TbTau95 associates with tRNA and U2 snRNA genes • Putative interacting partners of Tau95 might include some RNAP II regulators.

Effects of Leishmania major infection on the gut microbiome of resistant and susceptible mice.

Cutaneous leishmaniasis, a parasitic disease caused by Leishmania major, is a widely frequent form in humans. To explore the importance of the host gut microbiota and to investigate its changes during L. major infection, two different groups of mouse models were assessed. The microbiome of two parts of the host gut-ileum and colon-from infected and non-infected mice were characterised by sequencing of 16S rDNA using an Ion Torrent PGM platform. Microbiome analysis was performed to reveal changes related to the susceptibility and the genetics of mice strains in two different gut compartments and to compare the results between infected and non-infected mice. The results showed that Leishmania infection affects mainly the ileum microbiota, whereas the colon bacterial community was more stable. Different biomarkers were determined in the gut microbiota of infected resistant mice and infected susceptible mice using LEfSe analysis. Lactobacillaceae was associated with resistance in the colon microbiota of all resistant mice strains infected with L. major. Genes related to xenobiotic biodegradation and metabolism and amino acid metabolism were primarily enriched in the small intestine microbiome of resistant strains, while genes associated with carbohydrate metabolism and glycan biosynthesis and metabolism were most abundant in the gut microbiome of the infected susceptible mice. These results should improve our understanding of host-parasite interaction and provide important insights into the effect of leishmaniasis on the gut microbiota. Also, this study highlights the role of host genetic variation in shaping the diversity and composition of the gut microbiome. KEY POINTS: • Leishmaniasis may affect mainly the ileum microbiota while colon microbiota was more stable. • Biomarkers related with resistance or susceptibility were determined in the gut microbiota of mice. • Several pathways were predicted to be upregulated in the gut microbiota of resistant or susceptible mice.

Elucidating miR-146a-3p as a key player in autophagy and lipid metabolism in Leishmania major infection.

Autophagy is a key step involved in many unicellular eukaryotic diseases, including leishmaniasis, for cellular remodelling and differentiation during parasite's lifecycle. Lipids play a significant role in the infection process that begins with Leishmania major invading host cells. MicroRNAs (miRNAs), a family of small, 22-24 nucleotide noncoding regulatory RNAs, target mRNAs to modify gene expression and, subsequently, proteome output may have a regulatory role in altering the host cell processes. We observed miR-146a-3p expression increases in a time-dependent manner post Leishmania major infection. Transfecting miR-146a-3p mimic increases the expression of ATG7, an autophagy gene that encodes an E1-like enzyme in two ubiquitin-like conjugation systems required for autophagosome progression. HPGD (15-hydroxyprostaglandin dehydrogenase) operates as an enzyme, converting prostaglandin to its non-active form. Microarray data and western studies reveal that miR-146a-3p targets and inhibits HPGD, thereby increasing prostaglandin activity in lipid droplets. Herein, our research focuses on miR-146a-3p, which boosts ATG7 expression while reducing HPGD post Leishmania major infections helping us comprehend the intricate network of microRNA, autophagy, and lipid metabolism in leishmaniasis.

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

Molecular investigation of Leishmania in sandflies (Diptera: Psychodidae) and rodents (Mammalia: Rodentia) in Nahavand, west of Iran.

Cutaneous leishmaniasis caused by different species of Leishmania is transmitted by Phlebotominae sandflies. This disease remains a public health concern in Iran. Therefore, the present study aimed to examine Leishmania infection in sandflies and reservoir rodents in six rural regions of Nahavand, located in western Iran. From May to October 2022, sandflies and rodents were collected and identified at the species level. Additionally, rodents' skin lesions and earlobe specimens were collected separately for microscopic and molecular examination. All specimens were tested for Leishmania DNA by PCRs targeting the parasite's ITS-2 and 18S rRNA gene and positive were Sanger sequenced. A total of 3396 sandflies belonging to seven subgenera and 11 species, i.e., Phlebotomus papatasi (42.7%), P. major (20.6%), P. mascitti (0.3%), P. neglectus (0.2%), P. alexandri (0.2%), P. turanicus (0.3%), Sergentomyia murgabiensis (18.1%), S. dentata (10.5%), S. theodori (5.8%), S. antennata (1.1%), and S. pawlowski (0.1%) were identified. Based on the species population, 29 pools of sandflies were examined for the presence of Leishmania DNA using conventional PCR (cPCR), and individual DNAs were tested when positive. Leishmania major DNA was detected in two P. papatasi and Leishmania sp. in one P. major individual sandfly. This is the first report of Leishmania infection in sandflies from Hamadan province. The captured rodents (n = 61) belonged to four families and seven species, i.e., Arvicola amphibius (37.7%), Mus musculus (29.5%), Microtus socialis (13.1%), Apodemus sylvaticus (11.5%), Talpa davidiana (4.9%), Apodemus witherbyi (1.6%), and Rattus norvegicus (1.6%). Microscopic and molecular examinations of the rodent lesions and earlobes scored negative results. The presence of Leishmania in the Phlebotominae sandflies in Nahavand indicates a potential threat to humans and animals in the region. Regular monitoring and examination of the sandflies' population and timely diagnosis and treatment of new patients are strongly recommended.

Comparison of biosynthetic zinc oxide nanoparticle and glucantime cytotoxic effects on Leishmania major (MRHO/IR/75/ER).

Currently, zinc oxide (ZnO) particles are used in nanotechnology to destroy a wide range of microorganisms. Although pentavalent antimony compounds are used as antileishmanial drugs, they are associated with several limitations and side effects. Therefore, it is always desirable to try to find new and effective treatments. The aim of this research is to determine the antileishmanial effect of ZnO particles in comparison to the Antimoan Meglumine compound on promastigotes and amastigotes of Leishmania major (MRHO/IR/75/ER). After the extraction and purification of macrophages from the peritoneal cavity of C57BL/6 mice, L. major parasites were cultured in Roswell Park Memorial Institute-1640 culture medium containing fetal bovine serum (FBS) 10% and antibiotic. In this experimental study, the effect of different concentrations of nanoparticles was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric method, in comparison to the glucantime on promastigotes, amastigotes and healthy macrophages in the culture medium. The amount of light absorption of the obtained color from the regeneration of tetrazolium salt to the product color of formazan by the parasite was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the IC50 value was calculated. IC50 after 24 h of incubation was calculated as IC50 = 358.6 µg/mL. The results showed, that the efficacy of ZnO nanoparticles was favorable and dose-dependent. The concentration of 500 µg/mL of ZnO nanoparticles induced 84.67% apoptosis after 72. Also, the toxicity of nanoparticles was less than the drug. Nanoparticles exert their cytotoxic effects by inducing apoptosis. They can be suitable candidates in the pharmaceutical industry in the future.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

The effects of Leishmania RNA virus 2 (LRV2) on the virulence factors of L. major and pro-inflammatory biomarkers: an in vitro study on human monocyte cell line (THP-1).

BACKGROUND: Cutaneous Leishmaniasis (CL) is a parasitic disease with diverse outcomes. Clinical diversity is influenced by various factors such as Leishmania species and host genetic background. The role of Leishmania RNA virus (LRV), as an endosymbiont, is suggested to not only affect the pathogenesis of Leishmania, but also impact host immune responses. This study aimed to investigate the influence of LRV2 on the expression of a number of virulence factors (VFs) of Leishmania and pro-inflammatory biomarkers. MATERIALS AND METHODS: Sample were obtained from CL patients from Golestan province. Leishmania species were identified by PCR (LIN 4, 17), and the presence of LRV2 was checked using the semi-nested PCR (RdRp gene). Human monocyte cell line (THP-1) was treated with three isolates of L. major with LRV2 and one isolate of L. major without LRV2. The treatments with four isolates were administered for the time points: zero, 12, 24, 36, and 48 h after co-infection. The expression levels of Leishmania VFs genes including GP63, HSP83, and MPI, as well as pro-inflammatory biomarkers genes including NLRP3, IL18, and IL1ß, were measured using quantitative real-time PCR. RESULTS: The expression of GP63, HSP83, and MPI revealed up-regulation in LRV2 + isolates compared to LRV2- isolates. The expression of the pro-inflammatory biomarkers including NLRP3, IL1ß, and IL18 genes in LRV2- were higher than LRV2 + isolates. CONCLUSION: This finding suggests that LRV2 + may have a probable effect on the Leishmania VFs and pro-inflammatory biomarkers in the human macrophage model.

Revisiting epidemiology of leishmaniasis in central Asia: lessons learnt.

In this work we reviewed historical and recent data on Leishmania spp. infection combining data collected in Turkmenistan, Uzbekistan, Kazakhstan, Kyrgyzstan, Iran, China and Mongolia. We specifically focused on a complex of co-existing species (Leishmania major, Leishmania turanica and Leishmania gerbilli) sharing the same animal reservoirs and vectors. In addition, we analysed the presence of dsRNA viruses in these species and discussed future research directions to identify species-specific traits, which may determine susceptibility of different Leishmania spp. to viral infection.

Capparis spinosa inhibits Leishmania major growth through nitric oxide production in vitro and arginase inhibition in silico.

Cutaneous leishmaniasis is an infectious disease, considered as a major public health problem in different regions of the world. The current treatments are limited due to their toxicity and treatment failures, which have increased the search for new substances of natural origin to control this infection. Capparis spinosa is an important medicinal plant, rich in biochemical compounds with a broad range of activities including antimicrobial effects. Nevertheless, more investigations are still needed to determine its effect on Leishmania parasites. This study aimed to evaluate the effect of C. spinosa' extracts on Leishmania major promastigotes and amastigotes growth as well as on L-arginine metabolic pathways, especially the production of leishmanicidal molecules such as nitric oxide. Our results showed that C. spinosa' methanolic and aqueous extracts contained polyphenols and flavonoids at different concentrations. The methanolic extract of C. spinosa, compared to the aqueous extract, showed significantly higher amounts of total polyphenols (21.23 ± 1.08) mg GAE/g of dw (P < 0.05), as well as a higher antioxidant activity evaluated respectively by Reducing Power and DPPH (EC50: 0.31 ± 0.02 and 7.69 ± 1.28) mg/ml. Both extracts significantly inhibited L. major promastigotes and intra-macrophagic amastigotes growth in vitro in a dose-dependent manner (P < 0.001) and induced NO production not only in Leishmania-infected macrophages but also in uninfected macrophages, without showing any cytotoxicity in vitro. Furthermore, in silico docking studies showed that C. spinosa compounds identified by RP-HPLC exhibited inhibitory activity against the arginase enzyme. The leishmanicidal effect of C. spinosa may be due to its phenolic content and its mechanism of action may be mediated by an increase in NO production and by the inhibition of arginase enzyme in silico. These findings support the hypothesis that C. spinosa might be a valuable source of new biomolecules for leishmaniasis treatment.

Two New Cytotoxic Sesquiterpene-Amino Acid Conjugates and a Coumarin-Glucoside from Crossostephium chinense.

The Asteraceae family is a promising source of bioactive compounds, such as the famous Asteraceae plants Tanacetum cinerariifolium (pyrethrin) and Artemisia annua (artemisinin). As a result of our series of phytochemical studies of the subtropical plants, two novel sesquiterpenes, named crossoseamines A and B in this study (1 and 2, respectively), one undescribed coumarin-glucoside (3), and eighteen known compounds (4-21) were isolated from the aerial part of Crossostephium chinense (Asteraceae). The structures of isolated compounds were elucidated by spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY), IR spectrum, circular dichroism spectrum (CD), and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). All isolated compounds were evaluated for their cytotoxic activities against Leishmania major, Plasmodium falciparum, Trypanosoma brucei (gambiense and rhodesiense), and human lung cancer cell line A549 because of the high demand for the discovery of new drug leads to overcome the present side effects and emerging drug-resistant strains. As a result, the new compounds (1 and 2) showed significant activities against A549 (IC50, 1: 3.3 ± 0.3; 2: 12.3 ± 1.0 µg/mL), L. major (IC50, 1: 6.9 ± 0.6; 2: 24.9 ± 2.2 µg/mL), and P. falciparum (IC50, 1: 12.1 ± 1.1; 2: 15.6 ± 1.2 µg/mL).

New ψ-Santonin Derivatives from Crossostephium chinense and Their Anti-Proliferative Activities against Leishmania major and Human Cancer Cells A549.

Previously, we reported two cytotoxic ψ-santonin-amino acid conjugates isolated from the EtOAc layer of Crossostephium chinense. However, a further phytochemical investigation seems to be required because of the few reports of similar derivatives. In this study, we targeted the 1-BuOH layer, which resulted in the isolation of seven new ψ-santonin derivatives (1-7) together with ten known compounds (8-17). The structures of 1-7 were elucidated based on spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, and HMBC), IR spectrum, and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). The stereochemistry of new compounds was confirmed by NOESY and ECD calculations. All isolated compounds were evaluated by in vitro experiments for their anti-proliferative activities against Leishmania major, human lung cancer cell line A549, and Vero cells. As a result, most of the ψ-santonin derivatives, especially 1-5, showed significant cytotoxicity against L. major with a lower IC50 than the positive control we used (miltefosine).

In Silico and In Vitro Search for Dual Inhibitors of the Trypanosoma brucei and Leishmania major Pteridine Reductase 1 and Dihydrofolate Reductase.

The parasites Trypanosoma brucei (Tb) and Leishmania major (Lm) cause the tropical diseases sleeping sickness, nagana, and cutaneous leishmaniasis. Every year, millions of humans, as well as animals, living in tropical to subtropical climates fall victim to these illnesses' health threats. The parasites' frequent drug resistance and widely spread natural reservoirs heavily impede disease prevention and treatment. Due to pteridine auxotrophy, trypanosomatid parasites have developed a peculiar enzyme system consisting of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) to support cell survival. Extending our previous studies, we conducted a comparative study of the T. brucei (TbDHFR, TbPTR1) and L. major (LmDHFR, LmPTR1) enzymes to identify lead structures with a dual inhibitory effect. A pharmacophore-based in silico screening of three natural product databases (approximately 4880 compounds) was performed to preselect possible inhibitors. Building on the in silico results, the inhibitory potential of promising compounds was verified in vitro against the recombinant DHFR and PTR1 of both parasites using spectrophotometric enzyme assays. Twelve compounds were identified as dual inhibitors against the Tb enzymes (0.2 µM < IC50 < 85.1 µM) and ten against the respective Lm enzymes (0.6 µM < IC50 < 84.5 µM). These highly promising results may represent the starting point for the future development of new leads and drugs utilizing the trypanosomatid pteridine metabolism as a target.

Antileishmanial Activities of (Z)-2-(Nitroimidazolylmethylene)-3(2H)-Benzofuranones: Synthesis, In Vitro Assessment, and Bioactivation by NTR 1 and 2.

The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC50 values lower than 20 µM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC50 of 0.016 µM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.

Ligand-induced structural transitions combined with paramagnetic ions facilitate unambiguous NMR assignments of methyl groups in large proteins.

NMR spectroscopy allows the study of biomolecules in close-to-native conditions. Structural information can be inferred from the NMR spectra when an assignment is available. Protein assignment is usually a time-consuming task, being specially challenging in the case of large, supramolecular systems. Here, we present an extension of existing state-of-the-art strategies for methyl group assignment that partially overcomes signal overlapping and other difficulties associated to isolated methyl groups. Our approach exploits the ability of proteins to populate two or more conformational states, allowing for unique NOE restraints in each protein conformer. The method is compatible with automated assignment algorithms, granting assignments beyond the limits of a single protein state. The approach also benefits from long-range structural restraints obtained from metal-induced pseudocontact shifts (PCS) and paramagnetic relaxation enhancements (PREs). We illustrate the method with the complete assignment of the 199 methyl groups of a MILproSVproSAT methyl-labeled sample of the UDP-glucose pyrophosphorylase enzyme from Leishmania major (LmUGP). Protozoan parasites of the genus Leishmania causes Leishmaniasis, a neglected disease affecting over 12 million people worldwide. LmUGP is responsible for the de novo biosynthesis of uridine diphosphate-glucose, a precursor in the biosynthesis of the dense surface glycocalyx involved in parasite survival and infectivity. NMR experiments with LmUGP and related enzymes have the potential to unravel new insights in the host resistance mechanisms used by Leishmania major. Our efforts will help in the development of selective and efficient drugs against Leishmania.

Anti-leishmanial effects of resveratrol and resveratrol nanoemulsion on Leishmania major.

BACKGROUND: Leishmaniasis is a vector-borne disease that is endemic in the tropical and sub-tropical areas of the world. Low efficacy and high cytotoxicity of the current treatment regimens for leishmaniasis is one of the most important health problems. In this experimental study, anti-leishmanial effects of different concentrations of resveratrol and resveratrol nano-emulsion (RNE) were assessed. METHODS: RNE was prepared using the probe ultra-sonication method. The cytotoxicity was evaluated using the MTT technique on the L929 cell line. The anti-leishmanial activities on promastigotes of leishmania were assessed using vital staining and infected BALB/c mice were used to assess the in vivo anti-leishmanial effects. RESULTS: In vitro and in vivo assays revealed that all concentrations of resveratrol and RNE had valuable inhibitory effects against Leishmania major in comparison to the control group (P < 0.05). The half maximal inhibitory concentration (IC50) values were calculated as 16.23 and 35.71 µg/mL for resveratrol and RNE, respectively. Resveratrol and RNE showed no cytotoxicity against the L929 cell line. CONCLUSIONS: According to the potent in vitro and in vivo anti-leishmanial activity of RNE at low concentration against L. major, we suggest that it could be a promising anti-leishmanial therapeutic against L. major in the future.

Immunotherapeutic effects of Glycyrrhiza glabra and Glycyrrhizic Acid on Leishmania major-infection BALB/C mice.

Treatment of cutaneous leishmaniasis (CL) is a public health problem in endemic areas. The objective of the current study was to investigate the immunotherapeutic activities of the hydroalcoholic extract of Glycyrrhiza glabra (HEG) and glycyrrhizic acid (GA) in the treatment of Leishmania major (L. major)-infected BALB/c mice. In this study, the effect of HEG and GA was checked in vitro on growth of L. major promastigote and amastigote using MTT assay and microscopic counting, respectively. For in vivo experiment, the lesion induced by L. major on BALB/c mice were treated intraperitoneally with HEG, GA, meglumine antimoniate or phosphate buffer saline (negative control) for one month. Then, the lesion development and the parasite burden of the lymph node was assessed, the cytokine response (IFN-γ and IL-4) to Leishmania antigens was evaluated using ELISA method. The results showed that HEG and GA significantly inhibited the growth of L. major promastigotes and amastigotes, the lesion development, parasite burden in the lymph nodes, level of IFN-γ and the ratio of IFN-γ/IL-4 in HEG, GA and meglumine antimoniate-treated mice were significantly higher compared with the negative control group, there was no difference between the HEG, GA and meglumine antimoniate group. It is concluded that hydroalcoholic extract of G. glabra and glycyrrhizic acid showed therapeutic and immunomodulatory effects on L. major-infected BALB/c mice.