Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes

BACKGROUND: Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. RESULTS: A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. CONCLUSIONS: The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions

Bearing Capacity and Load-Displacement Behavior of Rigid Pads on Soft, Sensitive, Clay

The paper describes tests on rigid square pads at the UK national soft clay research site at Bothkennar, Scotland. The work was performed as a low cost adjunct to the instrumented pile research described by Lehane and Jardine (1992). Its aims were to investigate (i} bearing capacity, (ii) load-displacement response to short and long term loading, (iii) the applicability of relevant theories and (iv) relationships between soil properties determined in-situ and those measured in high quality laboratory tests

Development of a Novel Biological Intervertebral Disc Scaffold

Back pain is a major public health issue in our society, and is strongly correlated with the degeneration of intervertebral discs (IVDs). Current therapies are conservative or surgical, and no attempt to regenerate the IVD. The first goal of our project is to create a fully decellularized bovine caudal IVD to be used as a scaffold on which to seed adult human stem cells in an attempt to engineer a healthy, replacement IVD for patients suffering from IVD degeneration and lower back pain. The goal of decellularization is to eliminate DNA content while retaining glycosaminoglycan (GAG) content. Eliminating DNA content will prevent a foreign body response by the host\u27s immune system once the IVD is implanted. GAG is responsible for forming interfibrillar bridges with collagen fibrils and thus assisting in resisting compressive and tensile forces. By retaining GAG content in our decellularized IVDs we will maintain structural integrity of the extracellular matrix. The IVDs closest to the base of the bovine tail were targeted, because they are similar in size and biochemistry to the human IVD. We use a mix of conventional methods including freeze thaw, sonication and agitation in a solution of sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA). After decellularization, half of each IVD was placed in a tissue cassette and put formalin in preparation for histological analysis, and the other half was frozen prior to biochemical analysis (DMMB and PicoGreen assay). Our results thus far are promising in eliminating DNA content but show we have a large room for improvement in retaining GAG content

International Glossina Genome Initiative 2004-2014: a driver for post-genomic era research on the African continent

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a neglected disease that impacts 70 million people distributed over 1.55 million km2 in sub- Saharan Africa and includes at least 50% of the population of theDemocratic Republic of the Congo [1]. Trypanosoma brucei gambiense accounts for more than 98% of the infections in central and West Africa, the remaining infections being from Trypanosoma brucei rhodesiense in East Africa [2]. The parasites are transmitted to the hosts through the bite of an infected tsetse fly. Disease control is challenging as there are no vaccines, and effective, easily delivered drugs are still lacking. Treatment invariably involves lengthy hospitalization, with both medical and socioeconomic consequences.Web of Scienc

Assessing the functional performance of post-call hospital doctors using a Nintendo Wii.

Sleep deprivation is an established part of the working life for Non-Consultant Hospital Doctors (NCHDs) in Ireland. Concern exists about the effect of extended NCHD work hours. We utilised a Nintendo Wii to evaluate motor function of NCHDs both prior to their on-call shift and the day afterwards. Data was exported to SPSS ver. 15 for statistical analysis with p \u3c 0.05 considered significant. A total of 72 NCHDs were invited to participate in this study. There was a 62.5% (45) rate of follow-up. Overall 27 (60%) NCHDs were on medical call, with 18 (40%) on surgical call. There was no statistically significant difference between NCHDs pre-and post-call motor assessment scores. The majority of study participants (75.5%, n = 34) had four or more hours sleep. On-call duty allows for a greater than anticipated amount of sleep per on-call shift and therefore has a negligible effect on the motor skills of medical staff

Cryptic diversity within the major trypanosomiasis vector Glossina fuscipes revealed by molecular markers

Background: The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units. Principal Findings: The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f.fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled fromEthiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations. Conclusion/Significance: We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme

Prospects for developing odour baits to Control Glossina fuscipes spp., the major vector of human African trypanosomiasis

We are attempting to develop cost-effective control methods for the important vector of sleeping sickness, Glossina fuscipes spp. Responses of the tsetse flies Glossina fuscipes fuscipes (in Kenya) and G. f. quanzensis (in Democratic Republic of Congo) to natural host odours are reported. Arrangements of electric nets were used to assess the effect of cattle-, human- and pig-odour on (1) the numbers of tsetse attracted to the odour source and (2) the proportion of flies that landed on a black target (1 x 1 m). In addition responses to monitor lizard (Varanus niloticus) were assessed in Kenya. The effects of all four odours on the proportion of tsetse that entered a biconical trap were also determined. Sources of natural host odour were produced by placing live hosts in a tent or metal hut (volumes approximate to 16 m(3)) from which the air was exhausted at similar to 2000 L/min. Odours from cattle, pigs and humans had no significant effect on attraction of G. f. fuscipes but lizard odour doubled the catch (P<0.05). Similarly, mammalian odours had no significant effect on landing or trap entry whereas lizard odour increased these responses significantly: landing responses increased significantly by 22% for males and 10% for females; the increase in trap efficiency was relatively slight (5-10%) and not always significant. For G. f. quanzensis, only pig odour had a consistent effect, doubling the catch of females attracted to the source and increasing the landing response for females by similar to 15%. Dispensing CO2 at doses equivalent to natural hosts suggested that the response of G. f. fuscipes to lizard odour was not due to CO2. For G. f. quanzensis, pig odour and CO2 attracted similar numbers of tsetse, but CO2 had no material effect on the landing response. The results suggest that identifying kairomones present in lizard odour for G. f. fuscipes and pig odour for G. f. quanzensis may improve the performance of targets for controlling these species

Verapamil-Sensitive Transport of Quinacrine and Methylene Blue via the Plasmodium falciparum Chloroquine Resistance Transporter Reduces the Parasite's Susceptibility to these Tricyclic Drugs.

BACKGROUND: It is becoming increasingly apparent that certain mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) alter the parasite's susceptibility to diverse compounds. Here we investigated the interaction of PfCRT with 3 tricyclic compounds that have been used to treat malaria (quinacrine [QC] and methylene blue [MB]) or to study P. falciparum (acridine orange [AO]). METHODS: We measured the antiplasmodial activities of QC, MB, and AO against chloroquine-resistant and chloroquine-sensitive P. falciparum and determined whether QC and AO affect the accumulation and activity of chloroquine in these parasites. We also assessed the ability of mutant (PfCRT(Dd2)) and wild-type (PfCRT(D10)) variants of the protein to transport QC, MB, and AO when expressed at the surface of Xenopus laevis oocytes. RESULTS: Chloroquine resistance-conferring isoforms of PfCRT reduced the susceptibility of the parasite to QC, MB, and AO. In chloroquine-resistant (but not chloroquine-sensitive) parasites, AO and QC increased the parasite's accumulation of, and susceptibility to, chloroquine. All 3 compounds were shown to bind to PfCRT(Dd2), and the transport of QC and MB via this protein was saturable and inhibited by the chloroquine resistance-reverser verapamil. CONCLUSIONS: Our findings reveal that the PfCRT(Dd2)-mediated transport of tricyclic antimalarials reduces the parasite's susceptibility to these drugs

Tsetse control and Gambian sleeping sickness ; implications for control strategy

Background Gambian sleeping sickness (human African trypanosomiasis, HAT) outbreaks are brought under control by case detection and treatment although it is recognised that this typically only reaches about 75% of the population. Vector control is capable of completely interrupting HAT transmission but is not used because it is considered too expensive and difficult to organise in resource-poor settings. We conducted a full scale field trial of a refined vector control technology to determine its utility in control of Gambian HAT. Methods and Findings The major vector of Gambian HAT is the tsetse fly Glossina fuscipes which lives in the humid zone immediately adjacent to water bodies. From a series of preliminary trials we determined the number of tiny targets required to reduce G. fuscipes populations by more than 90%. Using these data for model calibration we predicted we needed a target density of 20 per linear km of river in riverine savannah to achieve >90% tsetse control. We then carried out a full scale, 500 km(2) field trial covering two HAT foci in Northern Uganda to determine the efficacy of tiny targets (overall target density 5.7/km(2)). In 12 months, tsetse populations declined by more than 90%. As a guide we used a published HAT transmission model and calculated that a 72% reduction in tsetse population is required to stop transmission in those settings. Interpretation The Ugandan census suggests population density in the HAT foci is approximately 500 per km2. The estimated cost for a single round of active case detection (excluding treatment), covering 80% of the population, is US$433,333 (WHO figures). One year of vector control organised within the country, which can completely stop HAT transmission, would cost US$42,700. The case for adding this method of vector control to case detection and treatment is strong. We outline how such a component could be organised

Discovery of spirooxadiazoline oxindoles with dual-stage antimalarial activity

© 2022 Published by Elsevier Masson SAS.Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na + efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold.This work was supported by FCT (Fundação para a Ciência e a Tecnologia, I.P.) through iMed.ULisboa (UID/DTP/04138/2019), project PTDC/QUI-QOR/29664/2017, and PhD fellowship SFRH/BD/137544/2018 (E. Lopes). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Financial support from FCT and Portugal 2020 to the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa – RNEM; LISBOA-01-0145-FEDER-402-022125) is also acknowledged.info:eu-repo/semantics/publishedVersio