•Enantioseparation of bicalutamide and its impurities was studied by HPLC on IA CSP.•Temperature effects on enantioselectivity and resolution were evaluated.•A ternary eluent system incorporating ...ethyl acetate was selected for mg-resolutions.•In these conditions, 10mg of the more active (R)-enantiomer of bicalutamide within 15min were isolated.
Direct HPLC separation of enantiomers of Bicalutamide (BCT), a non-steroidal antiandrogen used for the treatment of prostate cancer, was performed by using the immobilized amylose-based Chiralpak IA chiral stationary phase (CSP). Enantioselective conditions were achieved using standard normal phase mixtures n-hexane-alcohol (ethanol or 2-propanol) and a “non-standard” mobile phase containing ethyl acetate (EA). The chromatographic behaviour of the IA CSP under these elution modes was evaluated and compared at different temperatures. The eluent mixture n-hexane-EA-ethanol 100-30-5 (v/v/v) and the column temperature of 40°C were identified as the best operational conditions to carry out semipreparative enantioseparations on a 1-cm I.D. IA column. Using this protocol, about 960mg of (R)-BCT, which is the enantiomer with the almost entire anti-androgenic activity of BCT, per day could be isolated.
The analytical and semipreparative HPLC resolution of chiral impurities of BCT, and their empiric absolute configuration assignment by circular dichroism correlation method are also presented.
The neglected zoonosis cystic echinococcosis affects mainly pastoral and rural communities in both low-income and upper-middle-income countries. In Europe, it should be regarded as an orphan and rare ...disease. Although human cystic echinococcosis is a notifiable parasitic infectious disease in most European countries, in practice it is largely under-reported by national health systems. To fill this gap, we extracted data on the number, incidence, and trend of human cases in Europe through a systematic review approach, using both the scientific and grey literature and accounting for the period of publication from 1997 to 2021. The highest number of possible human cases at the national level was calculated from various data sources to generate a descriptive model of human cystic echinococcosis in Europe. We identified 64 745 human cystic echinococcosis cases from 40 European countries. The mean annual incidence from 1997 to 2020 throughout Europe was 0·64 cases per 100 000 people and in EU member states was 0·50 cases per 100 000 people. Based on incidence rates and trends detected in this study, the current epicentre of cystic echinococcosis in Europe is in the southeastern European countries, whereas historical endemic European Mediterranean countries have recorded a decrease in the number of cases over the time.
The FP7 project ’Human Cystic Echinococcosis ReseArch in CentraL and Eastern Societies’ (HERACLES), developed between 2013 and 2018 by nine partners in five countries, is one of the largest projects ...on cystic echinococcosis. Here we present the core HERACLES achievements, which should help to foster the translation of scientific investigations on health policies.
Display omitted
•The cluster G6/7 is a genotypic entity, distinct from G8 and G10, of Echinococcus canadensis.•Echinococcus canadensis G6/7 microdiversity revealed 73 mt cox1-nad1 concatenated ...haplotypes.•Echinococcus canadensis G6/7 haplotype grouping is region- and/or host-dependent.•Ancestral polymorphism and haplotypes of E. canadensis G6/7 were seen in sub-populations.
Among the genotype/species causing cystic echinococcosis, the taxonomic status of Echinococcus canadensis is only partially resolved. Within E. canadensis, four genotypes (G6, G7, G8 and G10) have been described based on short mitochondrial sequences, of which G6 and G7 (the ‘camel’ and the ‘pig’ strain, respectively) are closely related and variously regarded as microvariants of a single strain G6/7. Globally, this G6/7 cluster is the second most important agent of human cystic echinococcosis and is the predominant Echinococcus taxon in large parts of sub-Saharan Africa. To add data on the internal structure and the geographical distribution of this cluster, we analysed diversity and population structure of 296 isolates of E. canadensis from sub-Saharan Africa, the Middle East and Europe using the complete mitochondrial cytochrome c oxidase subunit 1 (cox1) (1,608bp) and NADH dehydrogenase subunit 1 (nad1) (894bp) gene sequences. Polymorphism of the mtDNA loci gave 51 (cox1), 33 (nad1) and 73 (cox1-nad1 concatenated) haplotypes. African and Middle Eastern isolates mainly grouped in a star-like structure around a predominant haplotype, while the European isolates produced more diversified networks. Although the cox1 diagnostic sequence for G6 is frequent in the African/Middle Eastern sub-cluster, and that for G7 is common in the European isolates, numerous intermediate variants prevent a clear distinction into ‘G6’ or ‘G7’, and the entire taxon is best treated as a common haplotype cluster G6/7. Meanwhile, the G6/7 cluster is clearly distinct from sequences of wildlife isolates of G8 and G10 from the northern hemisphere, and sequences of the latter genotypes were remarkably distant from each other. It is clear from the present study that, based on mitochondrial data, G6/7 is a coherent genotypic entity within E. canadensis that retains substantial intraspecific variance, and sub-populations share common ancestral polymorphisms and haplotypes. This study provides the basis for wider biogeographic comparison and population genetics studies of this taxon.
•Echinococcus canadensis was confirmed from pig and human hydatid isolates. E. granulosus (s.s.) was confirmed from sheep and cattle hydatid isolates.•The Serbian E. granulosus (s.s.) parsimony ...network was suggestive of bottleneck and/or balancing selection.•Our results appear to be consistent with the low genetic variation that characterizes marginal populations.
Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu stricto (s.s.) is a cosmopolitan zoonotic infection which is endemic in Serbia where it is subject to mandatory reporting. However, information on the incidence of the disease in humans and prevalence of hydatid infection in livestock remains limited. We used sequenced data of the cytochrome c oxidase subunit 1 (cox 1) mitochondrial gene to examine the genetic diversity and population structure of E. granulosus (s.s.) from intermediate hosts from Serbia. We also compared our generated nucleotide sequences with those reported for neighbouring European countries. Echinococcus canadensis was molecularly confirmed from pig and human hydatid isolates. E. granulosus (G1) was confirmed from sheep and cattle hydatid isolates as well as the first molecular confirmation in Serbia of E. granulosus G2 in sheep and E. granulosus G3 in sheep and cattle hydatid isolates. The Serbian E. granulosus (s.s.) parsimony network displayed 2 main haplotypes (SB02 and SB05) which together with the neutrality indices were suggestive of bottleneck and/or balancing selection. Haplotype analysis showed the presence of the common E. granulosus haplotype described from other worldwide regions. Investigation of the pairwise fixation (Fst) index suggested that Serbian populations of E. granulosus (s.s.) from sheep and cattle hosts showed moderate genetic differentiation. Six of the Serbian haplotypes (SB02-SB07) were shared with haplotypes from Bulgaria, Hungary and/or Romania. Further studies using a larger number of hydatid isolates from various locations across Serbia will provide more information on the genetic structure of E. granulosus (s.s.) within this region.
Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been ...described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity.
Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples.
All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay.
The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.
Hydatid cyst fluid (HCF)-based therapeutics has experimentally targeted approaches for treating human cancer cell lines. MicroRNA-365 (miR-365) has been reported to be an important tumor suppressor ...miRNA in cancers. However, it remains unknown, how miR-365 plays a pivotal role in inducing apoptosis in HCF-treated cancer cells in vitro. The fertile/infertile HCF was aspirated from liver of infected sheep and in terms of molecular taxonomy was identified as G1 genotype of Echinococcus granulosus sensu stricto. A375 human melanoma cancer cells were cultured into two groups: fertile and infertile HCF-treated A375 cells. To assess the cytotoxicity of various concentrations of HCF on melanoma cells, cell viability was determined by using MTT assay. The IC50 value of HCF on A375 cells was determined 85 μg/mL. Caspase-3 enzymatic activity was evaluated by fluorometric assay in the HCF-treated melanoma cells. In addition, the mRNA expression of Bax, Bcl-2, Caspase-9 and miR-365 were determined by qRT-PCR. Findings of MTT assay showed that concentrations 85 μg/mL to 100 μg/mL of fertile HCF have the highest mortality (50%–52%) on A375 cells during 24 h. The fold change of Bax/Bcl-2 ratio, Caspase-9, miR-365 and Caspase-3 activity was higher in the fertile HCF-treated melanoma cells compared to infertile fluid treated A375 cells and human normal epithelial cell (as control cell). In conclusion, we over-expressed the miR-365 in melanoma A375 cells, via treatment of fertile HCF. Our findings suggested that inducing high expression of miR-365 might be a negative regulator of melanoma growth through activation of pro-apoptotic Bax, Caspase-9 and Caspase-3 that are essential to intrinsic apoptotic pathway. These findings provide new insights into the use of Echinococcus HCF-derived metabolites in the design of drug therapies and in vivo tumor cell vaccine to combat melanoma progression.
Display omitted
•MicroRNA-365 promotes apoptosis in human melanoma cell A375 treated with hydatid cyst fluid•Inducing high expression of miR-365 might be a negative regulator of melanoma growth.•Intrinsic apoptotic pathway is activated by melanoma cell A375 treated with hydatid cyst fluid.
Rhinoviruses (RVs) have been linked to exacerbations of many pulmonary diseases, thus increasing morbidity and/or mortality in subjects at risk. Unfortunately, the wide variety of RV genotypes ...constitutes a major hindrance for the development of Rhinovirus replication inhibitors. In the current investigation, we have developed a novel series of pyrazole derivatives that potently inhibit the Rhinovirus replication. Compounds 10e and 10h behave as early stage inhibitors of Rhinovirus infection with a broad-spectrum activity against RV-A and RV-B species (EC50 < 0.1 μM). We also evaluate the dynamics of the emerging resistance of these promising compounds and their in vitro genotoxicity. Molecular docking experiments shed light on the pharmacophoric elements interacting with residues of the drug-binding pocket.
Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may ...expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC
) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.
Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu ...stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.
PDF
