Dihydrobenz[e][1,4]oxazepin-2(3H)-ones, a new anthelmintic chemotype immobilising whipworm and reducing infectivity in vivo

Trichuris trichiura is a human parasitic whipworm infecting around 500 million people globally, damaging the physical growth and educational performance of those infected. Current drug treatment options are limited and lack efficacy against the worm, preventing an eradication programme. It is therefore important to develop new treatments for trichuriasis. Using Trichuris muris, an established model for T. trichiura, we screened a library of 480 novel drug-like small molecules for compounds causing paralysis of the ex vivo adult parasite. We identified a class of dihydrobenz[e][1,4]oxazepin-2(3H)-one compounds with anthelmintic activity against T. muris. Further screening of structurally related compounds and resynthesis of the most potent molecules led to the identification of 20 active dihydrobenzoxazepinones, a class of molecule not previously implicated in nematode control. The most active immobilise adult T. muris with EC50 values around 25–50μM, comparable to the existing anthelmintic levamisole. The best compounds from this chemotype show low cytotoxicity against murine gut epithelial cells, demonstrating selectivity for the parasite. Developing a novel oral pharmaceutical treatment for a neglected disease and deploying it via mass drug administration is challenging. Interestingly, the dihydrobenzoxazepinone OX02983 reduces the ability of embryonated T. muris eggs to establish infection in the mouse host in vivo. Complementing the potential development of dihydrobenzoxazepinones as an oral anthelmintic, this supports an alternative strategy of developing a therapeutic that acts in the environment, perhaps via a spray, to interrupt the parasite lifecycle. Together these results show that the dihydrobenzoxazepinones are a new class of anthelmintic, active against both egg and adult stages of Trichuris parasites. They demonstrate encouraging selectivity for the parasite, and importantly show considerable scope for further optimisation to improve potency and pharmacokinetic properties with the aim of developing a clinical agent

Are mice good models for human neuromuscular disease? Comparing muscle excursions in walking between mice and humans

The mouse is one of the most widely used animal models to study neuromuscular diseases and test new therapeutic strategies. However, findings from successful pre-clinical studies using mouse models frequently fail to translate to humans due to various factors. Differences in muscle function between the two species could be crucial but often have been overlooked. The purpose of this study was to evaluate and compare muscle excursions in walking between mice and humans

Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti

Mosquito-borne viruses including dengue, Zika, and Chikungunya viruses, and parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe, and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. We identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that reduced A. aegypti larval motility. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC_{50} of 25.5 ± 5.0 µM and 22.3 ± 5.4 µM, respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to A. aegypti adults at 100 µM and 10 µM, and completely blocked egg laying when fed at 100 µM. Camptothecin and its derivatives are inhibitors of topoisomerase I, have known activity against several agricultural pests, and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries

Dynamic biospeckle analysis, a new tool for the fast screening of plant nematicide selectivity

Background: Plant feeding, free-living nematodes cause extensive damage to plant roots by direct feeding and, in the case of some trichodorid and longidorid species, through the transmission of viruses. Developing more environmentally friendly, target-specific nematicides is currently impeded by slow and laborious methods of toxicity testing. Here, we developed a bioactivity assay based on the dynamics of light 'speckle' generated by living cells and we demonstrate its application by assessing chemicals' toxicity to different nematode trophic groups.Results: Free-living nematode populations extracted from soil were exposed to methanol and phenyl isothiocyanate (PEITC). Biospeckle analysis revealed differing behavioural responses as a function of nematode feeding groups. Trichodorus nematodes were less sensitive than were bacterial feeding nematodes or non-trichodorid plant feeding nematodes. Following 24 h of exposure to PEITC, bioactivity significantly decreased for plant and bacterial feeders but not for Trichodorus nematodes. Decreases in movement for plant and bacterial feeders in the presence of PEITC also led to measurable changes to the morphology of biospeckle patterns.Conclusions: Biospeckle analysis can be used to accelerate the screening of nematode bioactivity, thereby providing a fast way of testing the specificity of potential nematicidal compounds. With nematodes' distinctive movement and activity levels being visible in the biospeckle pattern, the technique has potential to screen the behavioural responses of diverse trophic nematode communities. The method discriminates both behavioural responses, morphological traits and activity levels and hence could be used to assess the specificity of nematicidal compounds.</p

Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti

Mosquito-borne viruses including dengue, Zika, and Chikungunya viruses, and parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe, and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. We identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that reduced A. aegypti larval motility. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC_{50} of 25.5 ± 5.0 µM and 22.3 ± 5.4 µM, respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to A. aegypti adults at 100 µM and 10 µM, and completely blocked egg laying when fed at 100 µM. Camptothecin and its derivatives are inhibitors of topoisomerase I, have known activity against several agricultural pests, and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries

A transcriptomic snapshot of early molecular communication between Pasteuria penetrans and Meloidogyne incognita

© The Author(s). 2018Background: Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment. Results: A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle. Conclusions: Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.Peer reviewedFinal Published versio

Enhanced detection of threat materials by dark-field x-ray imaging combined with deep neural networks

X-ray imaging has been boosted by the introduction of phase-based methods. Detail visibility is enhanced in phase contrast images, and dark-field images are sensitive to inhomogeneities on a length scale below the system’s spatial resolution. Here we show that dark-field creates a texture which is characteristic of the imaged material, and that its combination with conventional attenuation leads to an improved discrimination of threat materials. We show that remaining ambiguities can be resolved by exploiting the different energy dependence of the dark-field and attenuation signals. Furthermore, we demonstrate that the dark-field texture is well-suited for identification through machine learning approaches through two proof-of-concept studies. In both cases, application of the same approaches to datasets from which the dark-field images were removed led to a clear degradation in performance. While the small scale of these studies means further research is required, results indicate potential for a combined use of dark-field and deep neural networks in security applications and beyond

C. elegans expressing D76N β_{2}-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β_{2} -m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β_{2} -m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β_{2} -m expressing worms. We also demonstrated the specificity of the β_{2} -m variant in determining the pathological phenotype by rescuing the wild type phenotype when β_{2} -m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

2,4-Diaminothieno[3,2-d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm

The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle

Azotobacter genomes: the genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412)

The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities