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 L. 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.

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.

Identification of a Proteasome-Targeting Arylsulfonamide with Potential for the Treatment of Chagas' Disease.

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between ß4 and ß5 subunits that catalyze chymotrypsin-like activity. A mutation in the ß5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the ß4/ß5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Sheep scab transmission: a spatially explicit dynamic metapopulation model.

Psoroptic mange (sheep scab), caused by the parasitic mite, Psoroptes ovis, is an important disease of sheep worldwide. It causes chronic animal welfare issues and economic losses. Eradication of scab has proved impossible in many sheep-rearing areas and recent reports of resistance to macrocyclic lactones, a key class of parasiticide, highlight the importance of improving approaches to scab management. To allow this, the current study aimed to develop a stochastic spatial metapopulation model for sheep scab transmission which can be adapted for use in any geographical region, exhibited here using data for Great Britain. The model uses agricultural survey and sheep movement data to geo-reference farms and capture realistic movement patterns. Reported data on sheep scab outbreaks from 1973 to 1991 were used for model fitting with Sequential Monte Carlo Approximate Bayesian Computation methods. The outbreak incidence predicted by the model was from the same statistical distribution as the reported outbreak data ([Formula: see text] = 115.3, p = 1) and the spatial location of sheep scab outbreaks predicted was positively correlated with the observed outbreak data by county ([Formula: see text] = 0.55, p < 0.001), confirming that the model developed is able to accurately capture the number of farms infected in a year, the seasonality of scab incidence and the spatial patterns seen in the data. This model gives insight into the transmission dynamics of sheep scab and will allow the exploration of more effective control strategies.

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.

Metabolic rate and resource depletion in the tick Ixodes ricinus in response to temperature.

Understanding the effects of temperature on the metabolic activity and the rate of depletion of energy reserves by Ixodes ricinus can represent an important contribution to explaining patterns of tick activity and the likely impacts of environmental change on tick and tick-borne disease risk. Here, a cohort of I. ricinus nymphs, males, and females was collected and placed into incubators at temperatures of between 5 and 30 °C. The protein, carbohydrate, total lipid, neutral lipid, and glycogen levels were measured for nymphs for up to 70 days and adults up to 42 days. In nymphs, at day 0, glycogen was the most abundant metabolite followed by carbohydrate, with relatively low concentrations of protein and lipids. For males, the concentrations of different metabolites were relatively similar. In contrast, for females, concentrations of glycogen and carbohydrate were relatively low compared to those of protein and neutral lipids. Significant exponential declines in metabolite concentrations of all metabolites were detected over time for all life-cycle stages and at all temperatures. Nymphs generally showed lower rates of resource depletion than adults at all temperatures. The lower thresholds for metabolic activity were estimated to be between -10 and -5 °C. The Q10 values, which describe the thermal sensitivity of metabolic rate, were estimated to be relatively low (1.5 for nymphs, 1.71 for males, and 1.63 for females) compared to insects where they are typically around 2.5 (range: 1.5-3), and this is considered to be an adaptation to increase survival during the extended inter-feed intervals.

Systematic analysis of Plasmodium myosins reveals differential expression, localisation, and function in invasive and proliferative parasite stages.

The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterized MyoA is a Class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite traversal, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL-B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito.

Determining the total energy budget of the tick Ixodes ricinus.

Precise and accessible techniques for measuring metabolic responses to environmental stress are essential to allow the likely impacts of climate and climate change on tick distribution, abundance and phenology to be predicted. A more detailed understanding of the metabolic profile of ticks may also help the complex responses to pathogen infection and effects on transmission to be evaluated. Here, a series of biochemical protocols employing spectrophotometric methods are used to determine the entire energy budget of ticks. Protein, carbohydrate, total lipid, neutral lipid and glycogen were measured in individual Ixodes ricinus nymphs and adults. Two key trends were identified: in adults, protein was relatively more abundant than in nymphs, whereas in nymphs, glycogen and carbohydrate were more abundant than in adults, with glycogen alone composing 39% of the mass of metabolites in nymphs compared to 15 and 10% in females and males, respectively. The methods used were able to successfully separate neutral lipids from the polar phospholipids and the importance of distinguishing stored from structural lipid in estimates of lipid reserves is emphasised. The results demonstrate that the spectrophotometric approaches deliver relatively rapid and reliable estimates of the total energetic budget and can be used to quantify the metabolic profiles of individual ticks, demonstrating their suitability for use in ecological and epidemiological studies.

Essential oils as tick repellents on clothing.

Essential oils show promise as natural alternatives to synthetic tick repellents, but few studies have investigated their repellent efficacy in vivo or under field conditions. Here, blanket-drags and standardised walks were employed to evaluate tick acquisition by 1 m2 cotton blankets or cotton trousers, respectively, in woodland edge habitats of known high tick abundance. Blankets and trousers had been treated with one of 5% oregano, rosemary, spearmint or thyme oils, 20% DEET (N,N-diethyl-3-methylbenzamide) (positive control) or ethanol excipient-only (negative control). The number of ticks present on the blankets or trousers differed significantly between treatments: spearmint oil treatments resulted in significantly fewer ticks than the negative controls for both blankets and trousers and significantly fewer ticks were present on the oregano oil treated blankets. For ticks that did attach to the trousers, the rate of drop off within 3 min was significantly higher for trousers treated with spearmint oil or thyme oil than ethanol, oregano oil and rosemary oil. No reduction in repellence was detected over a 24 h period between treatment and testing. The results suggest that 5% oregano and spearmint oils exhibit potential as natural clothing repellents, with an effective equivalence to 20% DEET.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.

Antitrypanosomal 8-Hydroxy-Naphthyridines Are Chelators of Divalent Transition Metals.

The lack of information regarding the mechanisms of action (MoA) or specific molecular targets of phenotypically active compounds can prove a barrier to their development as chemotherapeutic agents. Here, we report the results of our orthogonal genetic, molecular, and biochemical studies to determine the MoA of a novel 7-substituted 8-hydroxy-1,6-naphthyridine (8-HNT) series that displays promising activity against Trypanosoma brucei and Leishmania donovani High-throughput loss-of-function genetic screens in T. brucei highlighted two probable zinc transporters associated with resistance to these compounds. These transporters localized to the parasite Golgi apparatus. Directed by these findings, the role of zinc and other divalent cations in the MoA of these compounds was investigated. 8-HNT compounds were found to directly deplete intracellular levels of Zn2+, while the addition of exogenous Zn2+ and Fe2+ reduced the potency of compounds from this series. Detailed biochemical analyses confirmed that 8-HNT compounds bind directly to a number of divalent cations, predominantly Zn2+, Fe2+, and Cu2+, forming 2:1 complexes with one of these cations. Collectively, our studies demonstrate transition metal depletion, due to chelation, as the MoA of the 8-HNT series of compounds. Strategies to improve the selectivity of 8-HNT compounds are discussed.

An Intraplantar Hypertonic Saline Assay in Mice for Rapid Screening of Analgesics.

BACKGROUND: Development of new analgesics is limited by shortcomings of existing preclinical screening assays such as wide variations in response, suitability for a narrow range of analgesics, and propensity to induce tissue damage. Our aim was to determine the feasibility of a new in vivo animal assay as an analgesic screen based on nociceptive responses (licking and biting) after intraplantar (i.pl.) injection of hypertonic saline (HS) in mice. METHODS: With approval from the Institutional Animal Care Committee, we conducted a randomized, investigator-blinded in vivo study in adult CD-1 mice. We first studied the concentration-response relationship, time course, and sex difference of animals' nociceptive responses to HS. Subsequently, we assessed the screening ability of the HS assay to detect a range of established analgesics belonging to different classes. Finally, we performed histopathologic studies to assess potential tissue damage. RESULTS: The response produced by i.pl. HS was greater and longer in female than in male mice. The responses to HS were concentration dependent with minimal variance. Ten percent HS evoked a maximal response within the first 5 minutes. Morphine dose-dependently attenuated animals' nociceptive responses (1-10 mg/kg intraperitoneally [i.p.]). The peripherally restricted µ-opioid receptor agonist, loperamide, reduced nociceptive responses when injected locally (30-100 µg/paw, i.pl.) but not systemically (1-10 mg/kg, i.p.). Acetylsalicylic acid (300 mg/kg, i.p.), naproxen (150 mg/kg, i.p), and acetaminophen (300 mg/kg, i.p.) all decreased nociceptive responses, as did i.pl. coinjections of lidocaine (0.003%-1%) with 10% HS. Histopathologic assessment revealed no tissue damage due to HS. CONCLUSIONS: The i.pl. HS assay is easily performed, rapidly detects standard analgesics, and produces minimal animal suffering without tissue damage. We propose this assay as a useful addition to the armamentarium of existing preclinical analgesic screens.

The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei.

The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1-3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1-3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1-3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1-3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1-3 loss-of-function is a mechanism of antifolate drug resistance.

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

Variations of isovaline structure related to activity in the formalin foot assay in mice.

Current centrally acting analgesics such as opioids are associated with adverse effects that limit their use and threaten patient safety. Isovaline is a novel prototype analgesic that produces peripheral antinociception in several pain models with little or no effect on the central nervous system. The aim of this study was to establish a preliminary structure-activity relationship for isovaline derivatives by assaying efficacy in the formalin foot assay and central adverse effect profile in mice. Selected compounds were tested using the formalin foot assay to determine efficacy in reducing formalin-induced behaviors. Of the compounds tested, R-isovaline, S-isovaline, and 1-amino-1-cyclobutanecarboxylic acid reduced nocifensive behavior in phase II of the assay. These effects occurred without affecting performance on the rotarod, indicating that the reduction in nocifensive behaviors was not due to sedation or motor incoordination. Modifications to isovaline that increased its steric size without a cyclobutane ring formation produced compounds with no activity in the formalin foot assay. These findings indicate that the conformational stability of isovaline or the ability to form a cyclobutane ring is necessary for activity in the formalin foot assay.

Seismic and experimental insights into eruption precursors at Volcán de Colima.

We combine geophysical and experimental observations to interpret preeruptive unrest at Volcán de Colima in 1998. 17,893 volcanic earthquakes were detected between 1 October and 31 December 1998, including 504 clusters. Using seismic ambient noise interferometry, we observe a drop in velocity prior to the eruption linked to damage accumulation during magma ascent. This is supported by experimental observations where static stress causes a velocity decrease prior to failure. Furthermore, we observe acoustic emission clusters during the experiments, with lower porosity samples producing higher numbers of repeaters. This behavior introduces tensile failure as an additional viable mechanism for clusters during magma ascent. The findings suggest that preeruptive magma ascent may be monitored to variable degrees of accuracy via descriptions of damage accumulation and associated seismic velocity changes.

Ixodes ventalloi: morphological and molecular support for species integrity.

Despite their medical and veterinary importance, some tick species are so poorly studied, that their role within pathogen vector transmission cycles is difficult to assess. The tick Ixodes ventalloi is one such species, and its biology and phylogenetic status remain an issue of debate. In the present study, specimens of adult I. ventalloi (n = 65 females; n = 31 males) infesting cats in the Lipari Island (Aeolian archipelago, Sicily, southern Italy) were characterized morphologically and molecularly, the latter based on mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes. The genetic data and phylogenetic analyses for both mitochondrial genes suggest the existence of two distinct genogroups. The ecological and epidemiological significance of the genetic structure within the I. ventalloi endemic population remains to be determined. The results highlight the need for further analysis of this tick species, including whole mitochondrial genome sequencing and crossbreeding studies, which will be pivotal to complement features of its status as a vector of pathogens.

Traumatic Myiasis: A Neglected Disease in a Changing World.

Traumatic myiasis, the parasitic infestation by fly larvae in traumatic lesions of the tissues of living vertebrates, is a serious medical condition in humans and a welfare and economic issue in domestic animals. New molecular studies are providing insights into its evolution and epidemiology. Nevertheless, its incidence in humans is generally underreported, particularly in tropical and subtropical regions. Myiasis in domestic animals has been studied more extensively, but continuous management is difficult and expensive. A key concern is the inadvertent introduction and global spread of agents of myiasis into nonendemic areas, facilitated by climate change and global transport. The incursion of the New World screwworm fly (Cochliomyia hominivorax) into Libya is the most notable of many such range shifts and demonstrates the potential risks of these parasites and the costs of removing them once established in a geographic area. Nevertheless, the insect agents of myiasis can be of societal benefit to forensic science and in medicine as an aid to wound treatment (larval therapy).

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain.

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.