Post-transcriptional reprogramming by thousands of mRNA untranslated regions in trypanosomes.

Although genome-wide polycistronic transcription places major emphasis on post-transcriptional controls in trypanosomatids, messenger RNA cis-regulatory untranslated regions (UTRs) have remained largely uncharacterised. Here, we describe a genome-scale massive parallel reporter assay coupled with 3'-UTR-seq profiling in the African trypanosome and identify thousands of regulatory UTRs. Increased translation efficiency was associated with dosage of adenine-rich poly-purine tracts (pPuTs). An independent assessment of native UTRs using machine learning based predictions confirmed the robust correspondence between pPuTs and positive control, as did an assessment of synthetic UTRs. Those 3'-UTRs associated with upregulated expression in bloodstream-stage cells were also enriched in uracil-rich poly-pyrimidine tracts, suggesting a mechanism for developmental activation through pPuT 'unmasking'. Thus, we describe a cis-regulatory UTR sequence 'code' that underpins gene expression control in the context of a constitutively transcribed genome. We conclude that thousands of UTRs post-transcriptionally reprogram gene expression profiles in trypanosomes.

ResMAP-a saturation mutagenesis platform enabling parallel profiling of target-specific resistance-conferring mutations in Plasmodium.

New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the PfKRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing PfKRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with PfKRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed. IMPORTANCE: An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Screening of this high-coverage library with characterized PfKRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.

Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

Hotspot sheep scab management: A community-based regional approach in England.

BACKGROUND: Ovine psoroptic mange (sheep scab), caused by an infestation of the mite Psoroptes ovis, leads to clinical disease, economic loss and severely compromised animal welfare. Here, a community-based approach to the management of scab in three high-risk areas of England is described. METHODS: For each of the 254 farms included in the study, an initial survey of their clinical sheep scab history was followed up by a blood test (ELISA) to detect the presence of antibodies to P. ovis. This facilitated the coordination of treatment across groups of farms in each region. Blood testing was then repeated at the end of the treatment programme. RESULTS: On the first blood test in 2021/2022, 25.6% (±5.5%) of the flocks were positive for sheep scab. On the second test in 2022/2023, 9% (±3.94%) of the flocks tested were positive, showing a highly statistically significant reduction in prevalence overall, but with strong regional variation. LIMITATIONS: generating an understanding of the flock-level nature of the blood test and confidence in its detection of scab where clinical signs were not apparent provided ongoing challenges. CONCLUSIONS: The programme demonstrated that a focused community-based approach can be used to significantly reduce the prevalence of sheep scab in high-risk areas of England. The use of the blood test on all farms allowed the identification of subclinical sheep scab. The programme provides an effective model for sheep scab management on a national scale.

Short-course combination treatment for experimental chronic Chagas disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of people in the Americas and across the world, leading to considerable morbidity and mortality. Current treatment options, benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead to adverse side effects because of long treatment durations. Better treatment options are therefore urgently required. Here, we describe a pyrrolopyrimidine series, identified through phenotypic screening, that offers an opportunity to improve on current treatments. In vitro cell-based washout assays demonstrate that compounds in the series are incapable of killing all parasites; however, combining these pyrrolopyrimidines with a subefficacious dose of BNZ can clear all parasites in vitro after 5 days. These findings were replicated in a clinically predictive in vivo model of chronic Chagas disease, where 5 days of treatment with the combination was sufficient to prevent parasite relapse. Comprehensive mechanism of action studies, supported by ligand-structure modeling, show that compounds from this pyrrolopyrimidine series inhibit the Qi active site of T. cruzi cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Knowledge of the molecular target enabled a cascade of assays to be assembled to evaluate selectivity over the human cytochrome b homolog. As a result, a highly selective and efficacious lead compound was identified. The combination of our lead compound with BNZ rapidly clears T. cruzi parasites, both in vitro and in vivo, and shows great potential to overcome key issues associated with currently available treatments.

DNDI-6174 is a preclinical candidate for visceral leishmaniasis that targets the cytochrome bc1.

New drugs for visceral leishmaniasis that are safe, low cost, and adapted to the field are urgently required. Despite concerted efforts over the last several years, the number of new chemical entities that are suitable for clinical development for the treatment of Leishmania remains low. Here, we describe the discovery and preclinical development of DNDI-6174, an inhibitor of Leishmania cytochrome bc1 complex activity that originated from a phenotypically identified pyrrolopyrimidine series. This compound fulfills all target candidate profile criteria required for progression into preclinical development. In addition to good metabolic stability and pharmacokinetic properties, DNDI-6174 demonstrates potent in vitro activity against a variety of Leishmania species and can reduce parasite burden in animal models of infection, with the potential to approach sterile cure. No major flags were identified in preliminary safety studies, including an exploratory 14-day toxicology study in the rat. DNDI-6174 is a cytochrome bc1 complex inhibitor with acceptable development properties to enter preclinical development for visceral leishmaniasis.

RES-Seq-a barcoded library of drug-resistant Leishmania donovani allowing rapid assessment of cross-resistance and relative fitness.

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. New therapeutic options with diverse mechanisms of actions (MoAs) are required to consolidate progress toward control of this disease and combat drug resistance. Here, we describe the development of a scalable resistance library screen (RES-Seq) as a tool to facilitate the identification and prioritization of anti-leishmanial compounds acting via novel MoA. We have amassed a large collection of Leishmania donovani cell lines resistant to frontline drugs and compounds in the VL pipeline, with resistance-conferring mutations fully characterized. New phenotypic hits screened against this highly curated panel of resistant lines can determine cross-resistance and potentially shared MoA. The ability to efficiently identify compounds acting via previously established MoA is vital to maintain diversity within drug development portfolios. To expedite screening, short identifier DNA barcodes were introduced into resistant clones enabling pooling and simultaneous screening of multiple cell lines. Illumina sequencing of barcodes enables the growth kinetics and relative fitness of multiple cell lines under compound selection to be tracked. Optimal conditions allowing discrimination of resistant and sensitive clones were established (3× and 10× EC50 for 3 days) and applied to screening of a complex library with VL preclinical and clinical drug candidates. RES-Seq is set to play an important role in ensuring that anti-leishmanial compounds exploiting diverse mechanisms of action are developed, ultimately providing options for future drug combination strategies.IMPORTANCEVisceral leishmaniasis (VL) remains the third largest parasitic killer worldwide, responsible for 20,000-30,000 deaths each year. Control and ultimate elimination of VL will require a range of therapeutic options with diverse mechanisms of action to combat drug resistance. One approach to ensure that compounds in development exploit diverse mechanisms of action is to screen them against highly curated cell lines resistant to drugs already in the VL pipeline. The identification of cross-resistant cell lines indicates that test compounds are likely acting via previously established mechanisms. Current cross-resistance screens are limited by the requirement to profile individual resistant cell lines one at a time. Here, we introduce unique DNA barcodes into multiple resistant cell lines to facilitate parallel profiling. Utilizing the power of Illumina sequencing, growth kinetics and relative fitness under compound selection can be monitored revolutionizing our ability to identify and prioritize compounds acting via novel mechanisms.

Treatment of sheep prior to movement: its contribution to an effective scab (psoroptic mange) management strategy.

BACKGROUND: Ovine psoroptic mange (sheep scab) is an important disease of sheep worldwide caused by the parasitic mite, Psoroptes ovis. It has a negative impact on animal welfare and leads to significant economic losses for the sheep industry. Effective and targeted management is required to limit its transmission. METHODS: A stochastic metapopulation model of sheep scab transmission is used to investigate the contribution of the treatment of sheep prior to movements to sales, gatherings (predominantly markets) and away grazing to the reduction of prevalence of farms with scab in Great Britain. RESULTS: Treatment prior to movement to gatherings resulted in an 86% reduction in the overall prevalence of farms with scab and was more effective at reducing the overall prevalence of farms with scab than treatment before other categories of movements. The relative risk of farms having scab infection was inversely related to the percentage of farms which treated, but this relationship was not linear, with the biggest declines in the prevalence of farms with scab being achieved by small percentages of farms treating; a 50% relative reduction in the farm prevalence was achieved with only 15% of farms treating prior to gathering movements. CONCLUSIONS: The results suggest that pre-movement treatment of sheep could make an important contribution to national scab control and, in practice, the approach could be more highly targeted if used in conjunction with known geographic and management risk factors for scab.

Predicting the distribution of Ixodes ricinus and Dermacentor reticulatus in Europe: a comparison of climate niche modelling approaches.

BACKGROUND: The ticks Ixodes ricinus and Dermacentor reticulatus are two of the most important vectors in Europe. Climate niche modelling has been used in many studies to attempt to explain their distribution and to predict changes under a range of climate change scenarios. The aim of this study was to assess the ability of different climate niche modelling approaches to explain the known distribution of I. ricinus and D. reticulatus in Europe. METHODS: A series of climate niche models, using different combinations of input data, were constructed and assessed. Species occurrence records obtained from systematic literature searches and Global Biodiversity Information Facility data were thinned to different degrees to remove sampling spatial bias. Four sources of climate data were used: bioclimatic variables, WorldClim, TerraClimate and MODIS satellite-derived data. Eight different model training extents were examined and three modelling frameworks were used: maximum entropy, generalised additive models and random forest models. The results were validated through internal cross-validation, comparison with an external independent dataset and expert opinion. RESULTS: The performance metrics and predictive ability of the different modelling approaches varied significantly within and between each species. Different combinations were better able to define the distribution of each of the two species. However, no single approach was considered fully able to capture the known distribution of the species. When considering the mean of the performance metrics of internal and external validation, 24 models for I. ricinus and 11 models for D. reticulatus of the 96 constructed were considered adequate according to the following criteria: area under the receiver-operating characteristic curve > 0.7; true skill statistic > 0.4; Miller's calibration slope 0.25 above or below 1; Boyce index > 0.9; omission rate < 0.15. CONCLUSIONS: This comprehensive analysis suggests that there is no single 'best practice' climate modelling approach to account for the distribution of these tick species. This has important implications for attempts to predict climate-mediated impacts on future tick distribution. It is suggested here that climate variables alone are not sufficient; habitat type, host availability and anthropogenic impacts, not included in current modelling approaches, could contribute to determining tick presence or absence at the local or regional scale.

Development of a 2,4-Diaminothiazole Series for the Treatment of Human African Trypanosomiasis Highlights the Importance of Static-Cidal Screening of Analogues.

While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.

Ticks and tick-borne pathogens of domestic animals in Somalia and neighbouring regions of Ethiopia and Kenya.

Ticks and tick-borne pathogens (TBPs) of domestic animals in Somalia and neighbouring regions of Ethiopia and Kenya are reviewed to identify knowledge gaps in these regions, where unrestricted livestock movements across borders are common. Major scientific databases, such as PubMed, Web of Science, Scopus, CABI, and Google Scholar were searched, to retrieve articles based on papers published between 1960 and March 2023. Thirty-one tick species representing six genera (Rhipicephalus, Hyalomma, Amblyomma, Haemaphysalis, Ornithodoros and Argas) were reported to infest domestic animals, mainly livestock. Overall, the most represented species were Rhipicephalus pulchellus (up to 60% of specimens identified), followed by Hyalomma dromedarii (up to 57%), Hyalomma truncatum (up to 57%), Amblyomma lepidum (up to 21%), Amblyomma variegatum (up to 21%) and Amblyomma gemma (up to 19%), with morphological characterization being the principal method of tick identification. In addition, 18 TBPs, including zoonotic pathogens (e.g., Crimean-Congo haemorrhagic fever virus), were detected, with Babesia spp., Theileria spp., and Rickettsia spp. being the most commonly reported. Half of the pathogens documented were detected using molecular techniques, while the other half were detected by serology and microscopic techniques. Generally, ticks and TBPs in the region are under-studied, particularly, data relating to pet animals and equines is lacking. Further, the infection intensity and herd prevalence of ticks and TBPs is unclear because of insufficient data and poor approaches to quantitative analysis, making it difficult to propose management policies in the region. There is an urgent need, therefore, for more and better studies, particularly those that take a 'One Health' perspective, focusing on the prevalence and socioeconomic impact of ticks and TBPs in animals as well as in humans, so that sustainable control strategies against them can be planned.

Distribution of ticks in the Western Palearctic: an updated systematic review (2015-2021).

BACKGROUND: The distributions of ticks and tick-borne pathogens are thought to have changed rapidly over the last two decades, with their ranges expanding into new regions. This expansion has been driven by a range of environmental and socio-economic factors, including climate change. Spatial modelling is being increasingly used to track the current and future distributions of ticks and tick-borne pathogens and to assess the associated disease risk. However, such analysis is dependent on high-resolution occurrence data for each species. To facilitate such analysis, in this review we have compiled georeferenced tick locations in the Western Palearctic, with a resolution accuracy under 10 km, that were reported between 2015 and 2021 METHODS: The PubMed and Web of Science databases were searched for peer-reviewed papers documenting the distribution of ticks that were published between 2015 and 2021, using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The papers were then screened and excluded in accordance with the PRISMA flow chart. Coordinate-referenced tick locations along with information on identification and collection methods were extracted from each eligible publication. Spatial analysis was conducted using R software (version 4.1.2). RESULTS: From the 1491 papers identified during the initial search, 124 met the inclusion criteria, and from these, 2267 coordinate-referenced tick records from 33 tick species were included in the final dataset. Over 30% of articles did not record the tick location adequately to meet inclusion criteria, only providing a location name or general location. Among the tick records, Ixodes ricinus had the highest representation (55%), followed by Dermacentor reticulatus (22.1%) and Ixodes frontalis (4.8%). The majority of ticks were collected from vegetation, with only 19.1% collected from hosts. CONCLUSIONS: The data presented provides a collection of recent high-resolution, coordinate-referenced tick locations for use in spatial analyses, which in turn can be used in combination with previously collated datasets to analyse the changes in tick distribution and research in the Western Palearctic. In the future it is recommended that, where data privacy rules allow, high-resolution methods are routinely used by researchers to geolocate tick samples and ensure their work can be used to its full potential.

Sheep scab: comparison of spatial and temporal patterns determined by clinical diagnosis or ELISA.

BACKGROUND: Ovine psoroptic mange (sheep scab) is an infectious condition caused by an allergen-induced hypersensitivity response to the mite Psoroptes ovis. Infestation results in clinical disease, economic loss and welfare issues in many sheep-producing countries. The aim of this study was to compare the prevalence and spatial pattern of sheep scab on contiguous farms, using both self-reported clinical outbreak history (2012-2020) and serological testing with an enzyme-linked immunosorbent assay (2021/2022). METHODS: Farms included in the study were located in three regions of known high scab prevalence in North, Central and Southwest England. In total, 254 farms completed both a questionnaire, which provided the clinical scab history of the farm, and submitted results of serological testing with the ELISA. RESULTS: A scab outbreak was reported by 17.4% (± confidence interval [CI]: 4.6%; n = 48) of farms in 2020 based on clinical diagnosis; scab was diagnosed by the ELISA on 25.6% (± 5.5%; n = 65) of farms in 2021/2022. Comparison of self-reported clinical scab cases with the ELISA test results identified a group of farms (n = 52) that did not report scab in 2020, or in some cases did not report having scab over the previous 8 years (n = 20), but whose flocks were nevertheless seropositive in 2021/2022. CONCLUSION: A small number of flocks, particularly those using common grazings in North England, where handling is infrequent, often comprising less susceptible sheep breeds, may have persistent scab infestations that are generally undetected by clinical inspection. The data highlight the advantages of serological testing to identify exposure to scab in flocks where clinical signs are less easily detected.

Assessing the effectiveness of prophylactic treatment strategies for sheep scab.

Ovine psoroptic mange (sheep scab) is a condition caused by a hypersensitivity response to the ectoparasitic mite, Psoroptes ovis. It is an animal welfare concern and causes extensive economic losses to the sheep industry worldwide. More effective scab management is required to limit increases in infection prevalence, particularly given growing concerns over acaricide resistance. Here, a stochastic metapopulation model is used to explore the effectiveness of a range of prophylactic acaricide treatment strategies in comparison to no intervention. Over a simulated one-year period, movement control, based on the prophylactic treatment of animals being moved in sales, followed by farm biosecurity of bought in animals, was shown to be the most effective at reducing scab risk and more cost-effective than no intervention. Localised targeting of prophylaxis in areas of high scab prevalence was more effective than using prophylaxis at random, however, this localised effect declined post-treatment because of the import of infected animals. The analysis highlights the role of the movement of infected animals in maintaining high levels of scab infection and the importance of reducing this route of transmission to allow localised management to be effective.

Predicting the current and future risk of ticks on livestock farms in Britain using random forest models.

The most abundant tick species in northern Europe, Ixodes ricinus, transmits a range of pathogens that cause disease in livestock. As I. ricinus distribution is influenced by climate, tick-borne disease risk is expected to change in the future. The aims of this work were to build a spatial model to predict current and future risk of ticks on livestock farms across Britain. Variables relating both to tick hazard and livestock exposure were included, to capture a niche which may be missed by broader scale models. A random forest machine learning model was used due to its ability to cope with correlated variables and interactions. Data on tick presence and absence on sheep and cattle farms was obtained from a retrospective questionnaire survey of 926 farmers. The ROC of the final model was 0.80. The model outputs matched observed patterns of tick distribution, with areas of highest tick risk in southwest and northwest England, Wales, and west Scotland. Overall, the probability of tick presence on livestock farms was predicted to increase by 5-7 % across Britain under future climate scenarios. The predicted increase is greater at higher altitudes and latitudes, further increasing the risk of tick-borne disease on farms in these areas.

Prevalence of Babesia spp. pathogens in the ticks Dermacentor reticulatus and Ixodes ricinus in the UK.

The emergence of Babesia pathogens novel to the UK is of growing concern; these include Babesia canis and Babesia caballi. However, a better understanding of changes in the prevalence of endemic Babesia species such as Babesia venatorum and Babesia divergens is also of importance. Here, the prevalence of Babesia pathogens in both Dermacentor reticulatus and Ixodes ricinus ticks was assessed. Dermacentor reticulatus were collected from six sites known to harbour populations of this species in west Wales and southern England. DNA was extracted from 879 individual ticks and subjected to PCR and sequence analysis. Seven Babesia species were detected in 7.5% of the ticks, including B. caballi (0.68%), B. bovis (1.7%), B. microti (1.02%), B. bigemina (0.34%), B. capreoli (0.34%), and one isolate of B. canis (0.34%). Two of the field sites with grazing equines present had ticks that were positive for B. caballi. For I. ricinus, up to 200 nymphs were collected from each of 24 cattle farms in south-west England. Nymphs were divided into 6 pools of 30 from each farm for DNA extraction, PCR, and sequencing. Samples from seven out of the 24 farms tested positive for Babesia, and most were positive for more than one species. Babesia divergens was identified from five farms, and three of these farms had two pooled samples positive for B. divergens, which given the low overall prevalence rate suggests that B. divergens may be highly clustered within the tick population. Most of the remaining positive samples were Babesia venatorum, demonstrating that this zoonotic pathogen is widespread in livestock habitats. The data suggest that B. canis is not yet widely prevalent in established D. reticulatus populations in the UK, but that there is a need to raise awareness of the risk of equine piroplasmosis in areas with endemic D. reticulatus foci, since B. caballi appears more widely established.

Toolkit of Approaches To Support Target-Focused Drug Discovery for Plasmodium falciparum Lysyl tRNA Synthetase.

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.

Non-canonical endogenous expression of voltage-gated sodium channel NaV 1.7 subtype by the TE671 rhabdomyosarcoma cell line.

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50  = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.

Repositioning of a Diaminothiazole Series Confirmed to Target the Cyclin-Dependent Kinase CRK12 for Use in the Treatment of African Animal Trypanosomiasis.

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.

Effect of Cardiac Catheterization Personnel On Time to Treatment for Myocardial Infarction.

PURPOSE: To evaluate the effect of physician and nonphysician cardiac catherization laboratory personnel on the treatment of myocardial infarction. METHODS: Admissions data from 4 Las Vegas, Nevada hospitals were analyzed via multivariate regression analysis to determine predictors of reperfusion times. The goal for reperfusion is a door-to-balloon time of less than 90 minutes. RESULTS: Prehospital ST-segment elevation myocardial infarction (STEMI) activation, cardiologist arrival time, lifesaving measures, door-to-electrocardiogram (ECG) time, time and day, critical diagnostic examinations, and door-to-first-medical-doctor time all significantly affected door-to-balloon time. However, cardiac catheterization laboratory (CCL) staff arrival time did not affect door-to-balloon time. DISCUSSION: This study confirms the well-established importance of prehospital ECG and STEMI protocol activation. The results also indicate the importance of cardiologist arrival time on reperfusion times as this explained a significant amount of the explained variance in door-to-balloon time. CCL team arrival time did not affect door-to-balloon time, dispelling a long-held belief that reducing the response time of the CCL team significantly reduced reperfusion times. CONCLUSION: Although cardiologist arrival time influenced door-to-balloon time, CCL staff arrival time did not. Programs to provide greater laboratory coverage might help improve reperfusion times as well as assist STEMI program coordinators in developing more efficient protocols.