Veterinary Biosciences - Research Publications
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ItemSynthesis, Characterization, and Biological Activity of Ferrocenyl Analogues of the Anthelmintic Drug Monepantel(AMER CHEMICAL SOC, 2016-10-10)There is major demand for the development of structurally new anti-infectives using innovative approaches to circumvent multidrug resistance in parasites. Herein, we describe the synthesis and characterization of ferrocenyl precursors and derivatives (2-8) of an anthelmintic drug, monepantel. All compounds were isolated as their racemates and characterized by 1H, 13C, and 19F NMR spectroscopy, mass spectrometry, and IR spectroscopy. The purity of individual compounds was confirmed by elemental microanalysis. The molecular structures of three of the organometallic compounds (5-7) were also established by X-ray crystallography. The biological activities of these compounds were then evaluated in vitro on various important eukaryotic parasites, including H. contortus, T. colubriformis, and D. immitis. The potencies against D. immitis (canine heartworm) of two compounds, a ferrocene-containing precursor (4) and the final ferrocene-based monepantel derivative (8), were shown to be moderate (EC50 = 3.70 Ī¼g/mL for 4 and 5.60 Ī¼g/mL for 8) and were comparable with those of the controls AAD85 (EC50 = 2.20 Ī¼g/mL) and a commercial drug, ivermectin (EC50 = 1.00-3.00 Ī¼g/mL). The assessment of the cytotoxicity using cancerous HeLa and noncancerous MRC-5 cell lines revealed that these compounds have moderate to low toxicities in mammalian cells, thereby showing selective activity on parasites.
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ItemCan New Digital Technologies Support Parasitolocy Teaching and Learning?(ELSEVIER SCI LTD, 2016-07)Traditionally, parasitology courses have mostly been taught face-to-face on campus, but now digital technologies offer opportunities for teaching and learning. Here, we give a perspective on how new technologies might be used through student-centred teaching approaches. First, a snapshot of recent trends in the higher education is provided; then, a brief account is given of how digital technologies [e.g., massive open online courses (MOOCs), flipped classroom (FC), games, quizzes, dedicated Facebook, and digital badges] might promote parasitology teaching and learning in digital learning environments. In our opinion, some of these digital technologies might be useful for competency-based, self-regulated, learner-centred teaching and learning in an online or blended teaching environment.
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ItemOrganometallic Derivatization of the Nematocidal Drug Monepantel Leads to Promising Antiparasitic Drug Candidates(WILEY-V C H VERLAG GMBH, 2016-11)The discovery of novel drugs against animal parasites is in high demand due to drug-resistance problems encountered around the world. Herein, the synthesis and characterization of 27 organic and organometallic derivatives of the recently launched nematocidal drug monepantel (ZolvixĀ® ) are described. The compounds were isolated as racemates and were characterized by 1 H, 13 C, and 19 Fā NMR spectroscopy, mass spectrometry, and IR spectroscopy, and their purity was verified by microanalysis. The molecular structures of nine compounds were confirmed by X-ray crystallography. The anthelmintic activity of the newly designed analogues was evaluated in vitro against the economically important parasites Haemonchus contortus and Trichostrongylus colubriformis. Moderate nematocidal activity was observed for nine of the 27 compounds. Three compounds were confirmed as potentiators of a known monepantel target, the ACR-23 ion channel. Production of reactive oxygen species may confer secondary activity to the organometallic analogues. Two compounds, namely, an organic precursor (3āa) and a cymantrene analogue (9āa), showed activities against microfilariae of Dirofilaria immitis in the low microgram per milliliter range.
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ItemThe complement of family M1 aminopeptidases of Haemonchus contortus - Biotechnological implications(PERGAMON-ELSEVIER SCIENCE LTD, 2016)Although substantial research has been focused on the 'hidden antigen' H11 of Haemonchus contortus as a vaccine against haemonchosis in small ruminants, little is know about this and related aminopeptidases. In the present article, we reviewed genomic and transcriptomic data sets to define, for the first time, the complement of aminopeptidases (designated Hc-AP-1 to Hc-AP-13) of the family M1 with homologues in Caenorhabditis elegans, characterised by zinc-binding (HEXXH) and exo-peptidase (GAMEN) motifs. The three previously published H11 isoforms (accession nos. X94187, FJ481146 and AJ249941) had most sequence similarity to Hc-AP-2 and Hc-AP-8, whereas unpublished isoforms (accession nos. AJ249942 and AJ311316) were both most similar to Hc-AP-3. The aminopeptidases characterised here had homologues in C. elegans. Hc-AP-1 to Hc-AP-8 were most similar in amino acid sequence (28-41%) to C. elegans T07F10.1; Hc-AP-9 and Hc-AP-10 to C. elegans PAM-1 (isoform b) (53-54% similar); Hc-AP-11 and Hc-AP-12 to C. elegans AC3.5 and Y67D8C.9 (26% and 50% similar, respectively); and Hc-AP-13 to C. elegans C42C1.11 and ZC416.6 (50-58% similar). Comparative analysis suggested that Hc-AP-1 to Hc-AP-8 play roles in digestion, metabolite excretion, neuropeptide processing and/or osmotic regulation, with Hc-AP-4 and Hc-AP-7 having male-specific functional roles. The analysis also indicated that Hc-AP-9 and Hc-AP-10 might be involved in the degradation of cyclin (B3) and required to complete meiosis. Hc-AP-11 represents a leucyl/cystinyl aminopeptidase, predicted to have metallopeptidase and zinc ion binding activity, whereas Hc-AP-12 likely encodes an aminopeptidase Q homologue also with these activities and a possible role in gonad function. Finally, Hc-AP-13 is predicted to encode an aminopeptidase AP-1 homologue of C. elegans with hydrolase activity, suggested to operate, possibly synergistically with a PEPT-1 ortholog, as an oligopeptide transporter in the gut for protein uptake and normal development and/or reproduction of the worm. An appraisal of structure-based amino acid sequence alignments revealed that all conceptually translated Hc-AP proteins, with the exception of Hc-AP-12, adopt a topology similar to those observed for the two subgroups of mammalian M1 aminopeptidases, which possess either three (I, II and IV) or four (I-IV) domains. In contrast, Hc-AP-12 lacks the N-terminal domain (I), but possesses a substantially expanded domain III. Although further work needs to be done to assess amino acid sequence conservation of the different aminopeptidases among individual worms within and among H. contortus populations, we hope that these insights will support future localisation, structural and functional studies of these molecules in H. contortus as well as facilitate future assessments of a recombinant subunit or cocktail vaccine against haemonchosis.
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ItemPractical and low cost whole-organism motility assay: A step-by-step protocol(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2016-02)Here, we provide a step-by-step protocol for a practical and low cost whole-organism assay for the screening of chemical compounds for activity against parasitic worms. This assay has considerable advantages over conventional methods, mainly in relation to ease of use, throughput, time and cost. It is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation, and should be applicable to many different parasites and other organisms commensurate with the size of wells in the microtiter plates used for phenotypic screening.
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ItemMolecular characterization of Theileria orientalis from cattle in Ethiopia(ELSEVIER GMBH, 2016)This study reports the first molecular characterization of Theileria orientalis in local breeds of cattle in Ethiopia. A conventional PCR utilizing major piroplasm surface protein (MPSP) gene and an established multiplexed tandem PCR (MT-PCR) were used to characterize T. orientalis and to assess the infection intensity, respectively. Of 232 blood samples tested, T. orientalis DNA was detected in only 2.2% of samples using conventional PCR; two genotypes buffeli (1.3%; 3/232) and type 5 (0.9%; 2/232) of T. orientalis were detected. Phylogenetic analysis revealed that the buffeli MPSP sequences from Ethiopia were closely related to those reported from Kenya, Sri Lanka and Myanmar, and type 5 sequences from Ethiopia grouped with those from Korea, Japan, Vietnam and Thailand. A higher number of samples (3.9%; 9/232) were test-positive by MT-PCR and four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected. The average intensity of infections with genotypes buffeli (DNA copy numbers 11,056) and type 5 (7508) were significantly higher (P<0.0001) than the pathogenic genotype ikeda (61 DNA copies). This first insight into T. orientalis from cattle in Ethiopia using MPSP gene provides a basis for future studies of T. orientalis in various agroclimatic zones and of the impact of oriental theilerosis on cattle in this and other countries of Africa.
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ItemAssessment of the nematocidal activity of metallocenyl analogues of monepantel(ROYAL SOC CHEMISTRY, 2016)In this study, we present the design, synthesis, characterization and biological evaluation of structurally new ferrocenyl and ruthenocenyl derivatives of the organic anthelmintic monepantel (ZolvixĀ®). All seven metallocenyl derivatives prepared (4a/b, 5a/b, 6a/b and 7) were isolated as racemates and characterized by 1H, 13C and 19F NMR spectroscopies, mass spectrometry, IR spectroscopy and elemental microanalysis. The molecular structures of four compounds (4a/b, 6a and 7) were further confirmed by X-ray crystallography. The biological activities of the organometallic intermediates (4a/b) and organometallic derivatives of monepantel (5a/b, 6a/b and 7) were evaluated in vitro using parasitic nematodes of major importance in livestock, namely Haemonchus contortus and Trichostrongylus colubriformis. Two ferrocenyl compounds (4a and 6a) showed nematocidal activity, while the analogous ruthenocenyl compounds (4b and 6b) were not active at the highest concentration tested (10 Ī¼g mL-1). In order to obtain insight into the difference in activity between ferrocenyl and ruthenocenyl derivatives, the potential of the compounds for reactive oxidative species (ROS) production in live cells was assessed. Interestingly, neither the ferrocenyl nor the ruthenocenyl compounds (4a/b and 6a/b) produced significant ROS in HeLa cells when checked after 22 h, potentially indicating a redox-independent activity of 4a and 6a on the parasites. The selectivity of the compounds on parasites was confirmed by investigating their cytotoxicity profiles. None of these compounds was toxic either to HeLa or MRC-5 cells. Thus, 4a and 6a could be considered as interesting leads for further development of new classes of anti-parasitic agents.
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ItemA perspective on genomic-guided anthelmintic discovery and repurposing using Haemonchus contortus(ELSEVIER SCIENCE BV, 2016-06)High-throughput molecular and computer technologies have become instrumental for systems biological explorations of parasites. Investigating the genomes and transcriptomes of different developmental stages of parasitic nematodes can provide insights into gene expression, regulation and function in the parasite, which is a significant step toward understanding their biology as well as host interactions and disease. This article covers aspects of a talk given at the MEEGID XII conference in Thailand in 2014. Here, we refer to recent studies of the genomes and transcriptomes of socioeconomically important parasitic nematodes of animals; provide an account of the barber's pole worm (Haemonchus contortus) and emerging drug resistance problems in this and related worms; we also propose a genomic-guided drug discovery and repurposing approach, involving the prediction of the druggable genome, prioritization of drug targets, screening of compound libraries against H. contortus and, briefly, a hit-to-lead optimization approach. We conclude by indicating prospects that molecular tool kits for nematodes provide to the scientific community for future comparative genomic, genetic, proteomic, metabolomic, evolutionary, biological, ecological and epidemiological investigations, and as a basis for biotechnological outcomes and translation.
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ItemSelenophene and thiophene-core estrogen receptor ligands that inhibit motility and development of parasitic stages of Haemonchus contortus(BMC, 2016-06-16)BACKGROUND: Parasitic worms represent a substantial disease burden in animals and humans worldwide. The control of parasitic roundworms (nematodes) relies heavily on the use of anthelmintic drugs. However, widespread drug resistance in nematodes seriously compromises the effectiveness of many anthelmintics around the world. Thus, there is a need to discover new drugs, with unique modes of action, against parasites. METHODS: Here, we synthesised and tested 74 selective estrogen receptor modulators (SERMs) for in vitro-activity on parasitic larvae of Haemonchus contortus (barber's pole worm), one of the most important nematode pathogens of small ruminants (including sheep and goats) and a key representative of one of the largest groups of parasitic nematodes (the Strongylida) of animals. We also studied the morphology of treated and untreated larvae using scanning electron microscopy (SEM), and assessed the agonistic/antagonistic activity of SERMs in a human embryonic kidney cell line using a luciferase reporter assay system. RESULTS: We identified three SERMs (one selenophene and two thiophene-core compounds) with potent inhibitory activities (at 3-25 Ī¼M) on the motility and development of parasitic stages of H. contortus. An SEM examination of treated H. contortus revealed considerable damage to the cuticle of fourth- but not exsheathed, third-stage larvae; this damage appeared to be consistent with that observed upon treatment with monepantel but not moxidectin (control compounds). CONCLUSION: The potency of the three SERMs compared favourably with commercially available anthelmintics, such that they warrant further assessment as nematocides. Future studies could focus on assessing the selectivity of these SERMs to parasites, characterising their target(s) and/or designing analogs that are parasite-specific.
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ItemCAP protein superfamily members in Toxocara canis(BMC, 2016-06-24)BACKGROUND: Proteins of the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 (CAP) superfamily are recognized or proposed to play roles in parasite development and reproduction, and in modulating host immune attack and infection processes. However, little is known about these proteins for most parasites. RESULTS: In the present study, we explored CAP proteins of Toxocara canis, a socioeconomically important zoonotic roundworm. To do this, we mined and curated transcriptomic and genomic data, predicted and curated full-length protein sequences (nā=ā28), conducted analyses of these data and studied the transcription of respective genes in different developmental stages of T. canis. In addition, based on information available for Caenorhabditis elegans, we inferred that selected genes (including lon-1, vap-1, vap-2, scl-1, scl-8 and scl-11 orthologs) of T. canis and their interaction partners likely play central roles in this parasite's development and/or reproduction via TGF-beta and/or insulin-like signaling pathways, or via host interactions. CONCLUSION: In conclusion, this study could provide a foundation to guide future studies of CAP proteins of T. canis and related parasites, and might assist in finding new interventions against diseases caused by these parasites.