Evaluating Leadership Behaviors and Patient Safety Competencies During a Timed Multiple-Patient Simulation.

A digital count-up clock was incorporated into a multiple-patient simulation that required nursing students to respond to laboratory values and administer medications in a timely fashion. This study utilized observational methodology to analyze student response times and leadership behaviors. Results indicated a count-up clock can be utilized to assess attainment of patient safety competencies.

Bloodstream form Trypanosoma brucei depend upon multiple metacaspases associated with RAB11-positive endosomes.

Trypanosoma brucei possesses five metacaspase genes. Of these, MCA2 and MCA3 are expressed only in the mammalian bloodstream form of the parasite, whereas MCA5 is expressed also in the insect procyclic form. Triple RNAi analysis showed MCA2, MCA3 and MCA5 to be essential in the bloodstream form, with parasites accumulating pre-cytokinesis.

Protein kinases as drug targets in trypanosomes and Leishmania.

Protein kinases represent promising drug targets for a number of human and animal diseases. The recent completion of the sequenced genomes of three human-infective trypanosomatid protozoa, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi, has allowed the kinome for each parasite to be defined as 179, 156 and 171 eukaryotic protein kinases respectively, that is about one third of the human complement. The analysis revealed that the trypanosomatids lack members of the receptor-linked or cytosolic tyrosine kinase families, but have an abundance of STE and CMGC family protein kinases likely to be involved in regulating cell cycle control, differentiation and response to stress during their complex life-cycles.

Energy generation in insect stages of Trypanosoma brucei: metabolism in flux.

The generation of energy in African trypanosomes is a subject of undoubted importance. In bloodstream-form organisms, substrate-level phosphorylation of glucose is sufficient to provide the energy needs of the parasite. The situation in procyclic-form trypanosomes is more complex.

Trypanosoma brucei MOB1 is required for accurate and efficient cytokinesis but not for exit from mitosis.

Two MOB1 genes, MOB1-A and MOB1-B, were identified in Trypanosoma brucei. MOB1-A of T. brucei was shown to form a complex with TbPK50, a functional homologue of the Schizosaccharomyces pombe protein kinase Orb6, and immune precipitated MOB1-A exhibited histone H1 protein kinase activity.

A potential role for ICP, a Leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite.

The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants overexpressing ICP secreted the inhibitor and showed markedly reduced virulence in mice.

A plethora of targets, a paucity of drugs: progress towards the development of novel chemotherapies for human African trypanosomiasis.

Human African trypanosomiasis is a major health problem in large regions of Africa. Current chemotherapeutic options are limited and far from ideal. A diverse range of drug targets has been identified and validated in trypanosomes.

Generation of Leishmania mutants lacking antibiotic resistance genes using a versatile hit-and-run targeting strategy.

The development of a method to create defined mutants of Leishmania parasites lacking foreign genes conferring resistance to antibiotics has both experimental and practical applications. Mutants deficient in specific virulence genes have potential as attenuated live vaccines, but these can only be of clinical relevance if the antibiotic resistance genes used for selection of the mutants are subsequently removed. In addition, the limited number of antibiotic resistance genes that can be used for genetic manipulation of Leishmania means that a system for recycling them for subsequent use would be highly beneficial when multiple genetic modifications are wanted.

The Trypanosoma brucei cyclin, CYC2, is required for cell cycle progression through G1 phase and for maintenance of procyclic form cell morphology.

CYC2 is an essential PHO80-like cyclin that forms a complex with the cdc2-related kinase CRK3 in Trypanosoma brucei. In both procyclic and bloodstream form T. brucei, knock-down of CYC2 by RNA interference (RNAi) led to an accumulation of cells in G(1) phase.

Enzymes involved in the biogenesis of the nematode cuticle.

Nematodes include species that are significant parasites of man, his domestic animals and crops, and cause chronic debilitating diseases in the developing world; such as lymphatic filariasis and river blindness caused by filarial species. Around one third of the World's population harbour parasitic nematodes; no vaccines exist for prevention of infection, limited effective drugs are available and drug resistance is an ever-increasing problem. A critical structure of the nematode is the protective cuticle, a collagen-rich extracellular matrix (ECM) that forms the exoskeleton, and is critical for viability.

Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo.

Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L.

Clan CD cysteine peptidases of parasitic protozoa.

Parasitic protozoa contain an abundance of cysteine peptidases that are crucial for a range of important biological processes. The most studied cysteine peptidases of parasitic protozoa belong to the group of papain-like enzymes known as clan CA. However, several more recently identified cysteine peptidases differ fundamentally from the clan CA enzymes and have been included together in clan CD.

Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8.

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides.

Cyclin-dependent kinase homologues of Plasmodium falciparum.

The intraerythrocytic asexual cycle of the malarial parasite is complex and atypical: during schizogony the parasite undergoes multiple rounds of DNA replication and asynchronous nuclear division without cytokinesis. This cell cycle deviates from the classical eukaryotic cell cycle model where, 'DNA replicates only once per cell cycle'. A clear understanding of the molecular switches that control this unusual developmental cycle would be of great interest, both in terms of fundamental Plasmodium biology and in terms of novel potential drug target identification.

Two pathways of homologous recombination in Trypanosoma brucei.

African trypanosomes are unicellular parasites that use DNA recombination to evade the mammalian immune response. They do this in a process called antigenic variation, in which the parasites periodically switch the expression of VSG genes that encode distinct Variant Surface Glycoprotein coats. Recombination is used to move new VSG genes into specialised bloodstream VSG transcription sites.

Characterisation of the QM gene of Trypanosoma brucei.

The QM protein has been reported to have roles in both tumour suppression and transcription factor regulation in vertebrate cells, and in ribosome stability in both yeast and mammals. The present study isolated the QM gene of Trypanosoma brucei and determined its sequence. Alignment with QM sequences from Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Homo sapiens revealed greater than 60% identity.

Processing and trafficking of Leishmania mexicana GP63. Analysis using GP18 mutants deficient in glycosylphosphatidylinositol protein anchoring.

GPI8 is a clan CD, family C13 cysteine protease and the catalytic core of the GPI-protein transamidase complex. In Leishmania mexicana, GPI8 is nonessential, and Deltagpi8 mutants lack the GPI-anchored metalloprotease GP63, which is the major surface protein of promastigotes. We have identified the active site histidine and cysteine residues of leishmanial GPI8 and generated Deltagpi8 lines expressing modified GPI8 proteins.

The stage-regulated expression of Leishmania mexicana CPB cysteine proteases is mediated by an intercistronic sequence element.

The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.

The population genetics of Trypanosoma brucei and the origin of human infectivity.

The African trypanosome, Trypanosoma brucei, is a zoonotic parasite transmitted by tsetse flies. Two of the three subspecies, T. brucei gambiense and T.

Uptake and mode of action of drugs used against sleeping sickness.

Sleeping sickness is resurgent in Africa. Adverse side-effects and drug-resistance are undermining the few drugs currently licensed for use against this disease, which is caused by parasitic protozoa of the Trypanosoma brucei group. Pentamidine and suramin are used before parasites become manifest in the central nervous system, after which the organic arsenical melarsoprol is used.

Processing and trafficking of cysteine proteases in Leishmania mexicana.

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)).

Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase.

The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 microg ml(-1) was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T.

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins.

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum.

Characterisation of the loci encoding the glutamic acid and alanine rich protein of Trypanosoma congolense.

We have characterised the organisation of genes encoding the glutamate and alanine rich protein (GARP) surface coat of the procyclic and epimastigote stages of Trypanosoma congolense in the tsetse fly. The GARP genes are arranged at two, possibly physically linked, loci, one of which exhibits allelic variation. One locus contains a single GARP gene, whilst both alleles of the other have a large tandem array of polycistronically transcribed GARP genes.

A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation.

Antigenic variation is an immune evasion strategy used by African trypanosomes, in which the parasites periodically switch the expression of VSG genes that encode their protective variant surface glycoprotein coat. Two main routes exist for VSG switching: changing the transcriptional status between an active and an inactive copy of the site of VSG expression, called the bloodstream VSG expression site, or recombination reactions that move silent VSGs or VSG copies into the actively transcribed expression site. Nothing is known about the proteins that control and catalyze these switching reactions.