Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei . We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium .

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.

Heavy metals naturally are non-biodegradable constituents of the earth’s crust that accumulate and persist indefinitely in the ecosystem as a result of both human and natural activities. Their contamination of vegetables remains an issue of public health interest due to the frequency, and quantity of consumption. The over exposure to these heavy metals continues to pose serious health threat globally. This study was aimed to assess the heavy metal contents of staple vegetables [Telfairia occidentalis, Amaranthus hybridus and Ocimum gratissimum] within Enugu metropolis; the leaves were screened for heavy metals [Arsenic As, Lead Pb, Cadmium Cd, Nickel Ni, Chromium Cr and Cobalt Co], by atomic absorption spectroscopy (AAS). Results (Mean±SD, mgkg-1 ) showed that Amaranthus hybridus: contained [Pb-0.109±0.350, Cr -0.161±0.004]; Ocimum gratissimum: [Ni-0.179±0.028, Cd-0.033±0.006, Cr-0.176±0.036], and Telfairia occidentalis: [Pb-0.153±0.139, Co-0.198±0.148]; of which some values were slightly above WHO/FAO standards. Although, the estimated daily intakes (EDIs) were below referenced tolerable daily intakes (TDIs). The hazard quotients (HQs) were below 1 (HQ<1), but As and Cd, were exceptions; while the hazard index (HI) values were all above 1 (HI>1). The slightly above standard references of some of these heavy metals, and HI>1 values in this study are a concern, as potential health risks may arise amidst the population over a period of time, therefore, there is need to eliminate the likely sources of the latent contamination.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

Urogenital schistosomiasis is a neglected tropical disease caused by the parasite Schistosoma haematobium, which resides in the vasculature surrounding the urogenital system. Previous work has suggested that helminthic infections can affect the intestinal microbiome, and we hypothesized that S. haematobium infection could result in an alteration of immune system-microbiota homeostasis and impact the composition of the gut microbiota. To address this question, we compared the fecal microbiomes of infected and uninfected schoolchildren from the Argungu Local Government Area of Kebbi State, Nigeria, detecting significant differences in community composition between the two groups. Most remarkably, we observed a decreased abundance of Firmicutes and increased abundance of Proteobacteria - a shift in community structure which has been previously associated with dysbiosis. More specifically, we detected a number of changes in lower taxa reminiscent of inflammation-associated dysbiosis, including decreases in Clostridiales and increases in Moraxellaceae, Veillonellaceae, Pasteurellaceae, and Desulfovibrionaceae. Functional potential analysis also revealed an enrichment in orthologs of urease, which has been linked to dysbiosis and inflammation. Overall, our analysis indicates that S. haematobium infection is associated with perturbations in the gut microbiota and may point to microbiome disruption as an additional consequence of schistosome infection.

The recent massive reduction in the numbers of fresh Human African Trypanosomiasis (HAT) infection has presented an opportunity for the global elimination of this disease. To prevent a possible resurgence, as was the case after the reduced transmission of the 1960s, surveillance needs to be sustained and the necessary tools for detection and treatment of cases need to be made available at the points of care. In this review, we examine the available resources and make recommendations for improvement to ensure the sustenance of the already achieved gains to keep the trend moving towards elimination.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1.

Schistosomiasis is a debilitating disease affecting over 200 million people, with the highest burden of morbidity and mortality in African countries. Despite its huge impact on the health and socio-economic burden of the society, it remains a neglected tropical disease, with limited attention from governments and stakeholders in healthcare. One of the critical areas that is hugely under-developed is the development of accurate diagnostics for both intestinal and urogenital schistosomiasis. Diagnosis of schistosomiasis is important for the detection and treatment of disease in endemic and non-endemic settings. A conclusive detection method is also an indispensable part of treatment, both in the clinic and during mass drug administration (MDA), for the monitoring efficacy of treatment. Here, we review the available diagnostic methods and discuss the challenges encountered in diagnosis in resource limited settings. We also present the available diagnostics and cost implications for deployment in resource limited settings. Lastly, we emphasize the need for more funding directed towards the development of affordable diagnostic tools that is affordable for endemic countries as we work towards the elimination of the disease.

This study set out to evaluate self-medicated antibiotics and knowledge of antibiotic resistance among undergraduate students and community members in northern Nigeria. Antibiotic consumption pattern, source of prescription, illnesses commonly treated, attitude towards antibiotics, and knowledge of antibiotic resistance were explored using a structured questionnaire. Responses were analyzed and summarized using descriptive statistics. Of the 1230 respondents from undergraduate students and community members, prescription of antibiotics by a physician was 33% and 57%, respectively, amongst undergraduate students and community members. We tested the respondents’ knowledge of antibiotic resistance (ABR) and found that undergraduate students displayed less knowledge that self-medication could lead to ABR (32.6% and 42.2% respectively). Self-medication with antibiotics is highly prevalent in Northwest Nigeria, with most medicines being purchased from un-licensed stores without prescription from a physician. We also observed a significant gap in respondents’ knowledge of ABR. There is an urgent need for public health authorities in Nigeria to enforce existing laws on antibiotics sales and enlighten the people on the dangers of ABR.

Typhoid fever causes significant morbidity and mortality in developing countries, with inaccurate estimates in some countries affected, especially those situated in Sub-Saharan Africa. Disease burden assessment is limited by lack of a high degree of sensitivity and specificity by many current rapid diagnostic tests. Some of the new technologies, such as PCR and proteomics, may also be useful but are difficult for low-resource settings to apply as point-of-care diagnostics. Weak laboratory surveillance systems may also contribute to the spread of multidrug resistant Salmonella serovar Typhi across endemic areas. In addition, most typhoid-endemic countries employ serological tests that have low sensitivity and specificity making diagnosis unreliable. Here we review currently available typhoid fever diagnostics, and advances in serodiagnosis of S. Typhi.

ETHNOPHARMACOLOGICAL RELEVANCE:Leaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. AIM OF THE STUDY:To investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. MATERIAL AND METHODS:Plants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. RESULTS:Seven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3β,13β-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. CONCLUSION:We have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species.

INTRODUCTION:Trypanosomes are protozoan parasites of vertebrates transmitted by blood-sucking tsetse fly. Trypanosomes remain a constant threat to the lives of humans and animals throughout large regions of Africa.AIMS AND OBJECTIVES:This study investigated the presence, prevalence, and species of trypanosome parasite in tsetse flies caught in two areas of no previous documented history of trypanosome infection.MATERIALS AND METHODS:For this purpose, 63 and 77 nonterenal tsetse flies were collected from Oji River and Emene areas of Enugu State Nigeria, respectively. Genomic DNA was isolated from the whole tsetse fly using genomic DNA extraction kit. Identification and characterization of trypanosome were done using two approaches: the amplification of internal transcribed spacer 1 of ribosomal DNA and the use of primers specific to Trypanozoon.RESULTS:In Oji River, of 63 tsetse flies collected, the identification of trypanosome parasite was done on 57 flies and 6 (10.71%) tsetse flies were infected with trypanosome parasite. Six flies were infected with Trypanosoma Congolense, 2 with Trypanosoma Vivax, and 1 with Trypanosoma brucei. Two mixed infections of T. vivax and T. congolense and 1 mixed infection of T. brucei and T. congolense was also identified. In Emene, of 77 tsetse flies collected, the identification of trypanosome parasite was done on 66 flies and 11 (16.6%) tsetse flies were infected with trypanosome parasite. Nine flies were infected with T. congolense, 2 with T. vivax, and 3 with T. brucei. Mixed infections identified include 2 mixed infections of T. brucei and T. congolense and 1 mixed infections of T. vivax and T. brucei. None of the subspecies of T. brucei were detected using species specific primers.DISCUSSION:This study shows the parasitological evidence on the occurrence of animal African trypanosomiasis and also demonstrated that there is likely no active transmission of human African trypanosomiasis in the study areas.CONCLUSION:This study shows that there is likely no active transmission of human African trypanosomiasis going on in these localities since no human infective form of the parasite was detected.

Background Amlodipine and hydrochlorothiazide (HCTZ) are commonly prescribed in Nigeria either as a monotherapy or in combination with other drugs. The present study was designed to investigate the antihypertensive efficacy of monotherapy with amlodipine or HCTZ and their effects on electrolyte profile in patients with mild to moderate hypertension. Methods A single-blind randomized clinical study was used; fifty patients newly diagnosed with mild to moderate hypertension (aged 33 to 60 years) were recruited and divided into two groups: amlodipine or hydrochlorothiazide each comprising of 25 subjects. The subjects received 5mg of amlodipine or 25mg of hydrochlorothiazide in their respective group once daily for 4 weeks. Blood pressure, serum and urine electrolytes were measured at baseline and weekly throughout the experiment. Results At the end of follow up, amlodipine reduced systolic and diastolic blood pressure significantly more (p<0.001) than HCTZ. At the end of follow up, blood pressure was reduced to normal in 80% of the subjects in amlodipine group compared to 50% in HCTZ. Amlodipine had no significant effect on electrolyte profile of subjects unlike HCTZ which significantly changed both their serum and urine electrolytes. Conclusions Monotherapy with amlodipine was more effective than HCTZ in black patients with mild to moderate hypertension and in addition maintained electrolyte balance.

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [H-3]-uridine but not of [H-3]-inosine. Conversely, the NT2-like genes mediated uptake of [H-3]-inosine but not [H-3]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species essential information for the development of pyrimidine-based chemotherapy. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.

BACKGROUND:Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. METHODOLOGY/PRINCIPAL FINDINGS:Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. CONCLUSIONS/SIGNIFICANCE:Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential.

SummaryThe Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1−/− T. brucei line. Out of a set of six reported SNPs from a reported ‘resistance allele’, none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1‐WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [3H]‐adenosine and [3H]‐diminazene, identical to the tbat1−/− control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [3H]‐diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

The effects of sub-chronic administration of aqueous extract of Hibiscus sabdariffa (HS) on liver and kidney functions were investigated in Wistar rats. Thirty Wistar rats were randomly divided into six groups of 5 rats each. Group A served as the control and was given 5ml/kg of normal saline. Groups B to F were administered HS extract at 100, 500, 1000, 2000 and 5000mg/kg respectively for 6 weeks. Indices of liver and kidney functions were measured and body weight changes monitored. A dose-dependent increase in the activities of liver enzymes and kidney indices was observed. The activities of aspartate aminotranferase, alanine aminotransferase and alkaline phosphatase increased significantly (p<0.05) at doses of 2000mg/kg and above when compared to control while urea, creatinine and uric acid increased significantly (p<0.05) at 1000mg/kg and above. There was a significant (p<0.05) and dose-dependent decrease in weights of rats in the experimental groups. Signs of renal impairment were observed at 1000mg/kg and above while hepatic impairment occurred at 2000mg/kg and above. Thus, the present study has shown that below 1000mg/kg, HS consumption is safe and will not affect the functions of the liver and kidney.

Treatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four new N-hydroxy and 12 new N-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluated in vitro against Trypanosoma brucei rhodesiense and in vivo in murine models of first-and second-stage sleeping sickness. Resistance profile, physicochemical parameters, in vitro BBB permeability, and microsomal stability also were determined. The N-hydroxy imidazoline analogues were the most effective in vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl) amino)-N-(4-((1-hydroxy-4,5-dihydro-1Himidazol- 2-yl) amino) phenyl) benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was found in vitro, suggesting that N-hydroxy imidazolines are metabolically stable and have intrinsic activity against T. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d in T. brucei was independent of known drug transporters (i.e., T. brucei AT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, the N-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.