Search

Background Infection with Trypanosoma cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of T. cruzi biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite. Methodology/Principal findings Here, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a T. cruzi reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the in vivo phenotype can be rapidly assessed. Conclusions/Significance The techniques described here will have multiple applications for studying aspects of T. cruzi biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a community resource and are freely available on request., Author summary 5–8 million people in Latin America are infected with the single-cell parasite Trypanosoma cruzi, the causative agent of Chagas disease. Of these, approximately one-third will develop chronic disease pathology, leading to disability and premature death. Only two drugs are available, both of which can have severe side effects. We know relatively little about the chronic phase of this infection because the parasite burden is extremely low, and animal models have limitations. Here, we describe a T. cruzi reporter strain that has been genetically modified to express a fusion protein which is both bioluminescent and fluorescent. These parasites can be monitored throughout the infection, and individual parasites identified in tissue sections from infected mice. This allows us for the first time to analyse host-parasite interactions at a cellular level in the chronic phase of infection. We have also incorporated a streamlined version of the CRISPR/Cas9 genome editing system into the reporter strain. We demonstrated the utility of this system by generating null mutants in the flagellar attachment protein GP72, by replacing the green fluorescent protein with a red fluorescent protein, and by tagging the endogenous protein DNA topoisomerase 1A. This T. cruzi reporter line will significantly enhance our ability to unravel the pathogenesis and immunology of chronic Chagas disease.

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In $\textit{Trypanosoma brucei}$ and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.

The expression of transgenes encoding proteins modified to contain residues that impart a particular property, ‘tagged proteins’, is central to the post-genomic analysis of any organism. Trypanosoma brucei is a model kinetoplastid protozoan pathogen and has the most advanced repertoire of tools for reverse genetic analysis available for any protozoan. The vast majority of these tools take advantage of the predominance of homologous over non-homologous recombination to target constructs to specific genomic loci. Initially the targeting was used to direct unregulated transgenes to transcribed regions of the genome [1] and to perform gene deletions [2,3]. A leap forward in the sophistication of reverse genetic experiments occurred with the development of trypanosome cell lines expressing the tetracycline repressor (TetR) protein which facilitated tetracycline-regulated expression of transgenes [4,5]. Further development of cell lines expressing both TetR and bacteriophage T7 RNA polymerase (T7RNAP) allowed transgenes to be transcribed and expressed at very high levels [6]. The TetR- and T7RNAP-expressing cell lines are also central to most RNA interference-based analyses of gene function currently performed in trypanosomes [7–10]. In nearly all cases, an antibiotic resistance gene is used as the selectable marker. The DNA used for targetted integration is usually a linearised plasmid; targetting using PCR products directly is possible [11–13] but integration is less efficient and does not usually offer inducible expression. The expression of tagged proteins has become central to the technologies that have developed to analyse the function of individual genes. The tag can have a range of functions falling into two main categories: the first is to provide evidence for the sub-cellular localisation of a protein in living cells using a fluorescent protein tag (see, for example [14]) or in fixed cells using a fluorescent protein or epitope tag (see, for example [15]). The second is to facilitate the purification of complexes which, when allied with mass spectrometric analysis and knowledge of the genome sequence, allows the identification of components of multimeric proteins. Two types of tag have been developed successfully in yeast: (i) the tandem affinity tag where two successive rounds of purification are used [16], and (ii) a tandem epitope tag which is used in a single step purification [17]. To date, the former has been exploited more in trypanosomes [18,19]. In the future, the investigation of transient protein-protein interactions in vivo could be analysed by techniques such as fluorescence resonant energy transfer (FRET) which is dependent on tagging the two target proteins with different fluorescent proteins [20]. Here, we describe five sets of plasmids that represent a substantial collection of publicly available vectors for adding tags to the N- and C-termini of proteins in T. brucei.

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

Objectives: To estimate the economic efficiency of tight blood pressure control, with angiotensin converting enzyme inhibitors or beta blockers, compared with less tight control in hypertensive patients with type 2 diabetes. Design: Cost effectiveness analysis incorporating within trial analysis and estimation of impact on life expectancy through use of the within trial hazards of reaching a defined clinical end point. Use of resources driven by trial protocol and use of resources in standard clinical practice were both considered. Setting: 20 hospital based clinics in England, Scotland, and Northern Ireland. Subjects: 1148 hypertensive patients with type 2 diabetes from UK prospective diabetes study randomised to tight control of blood pressure (n=758) or less tight control (n=390). Main outcome measure: Cost effectiveness ratios based on (a) use of healthcare resources associated with tight control and less tight control and treatment of complications and (b) within trial time free from diabetes related end points, and life years gained. Results: Based on use of resources driven by trial protocol, the incremental cost effectiveness of tight control compared with less tight control was cost saving. Based on use of resources in standard clinical practice, incremental cost per extra year free from end points amounted to £1049 (costs and effects discounted at 6% per year) and £434 (costs discounted at 6% per year and effects not discounted). The incremental cost per life year gained was £720 (costs and effects discounted at 6% per year) and £291 (costs discounted at 6% per year and effects not discounted). Conclusions: Tight control of blood pressure in hypertensive patients with type 2 diabetes substantially reduced the cost of complications, increased the interval without complications and survival, and had a cost effectiveness ratio that compares favourably with many accepted healthcare programmes.

OBJECTIVE: To determine whether tight control of blood pressure prevents macrovascular and microvascular complications in patients with type 2 diabetes. DESIGN: Randomised controlled trial comparing tight control of blood pressure aiming at a blood pressure of

OBJECTIVE: To determine whether tight control of blood pressure with either a beta blocker or an angiotensin converting enzyme inhibitor has a specific advantage or disadvantage in preventing the macrovascular and microvascular complications of type 2 diabetes. DESIGN: Randomised controlled trial comparing an angiotensin converting enzyme inhibitor (captopril) with a beta blocker (atenolol) in patients with type 2 diabetes aiming at a blood pressure of =300 mg/l (5% and 9%). The proportion of patients with hypoglycaemic attacks was not different between groups, but mean weight gain in the atenolol group was greater (3.4 kg v 1.6 kg). CONCLUSION: Blood pressure lowering with captopril or atenolol was similarly effective in reducing the incidence of diabetic complications. This study provided no evidence that either drug has any specific beneficial or deleterious effect, suggesting that blood pressure reduction in itself may be more important than the treatment used.

The successful development of a long-term organ culture system has made it possible to perform experiments on rat colonic mucosa in vitro. However, the effect of trauma or the withdrawal of trophic influences in culture may result in the disturbance of proliferation within the tissue. In this paper we describe an investigation designed to characterise the culture system by a comparison of the proliferative parameters in vitro with those in vivo. Stathmokinetic experiments were performed in vivo and in vitro to estimate cell birth rate. Mitotic indices were also calculated. The in vivo birth rate (7.8 +/- 0.8 cells/1000 cells/hour) and the in vitro birth rate for the whole explant (7.7 +/- 0.5 cells/1000 cells/hour) were found to be similar. A study of crypts in the centre and at the edge of the cultured explants, however, indicated that proliferation at the two sites differed markedly, the birth rate at the edge (7.5 +/- 0.9 cells/1000 cells/hour) being approximately twice that at the centre (3.2 +/- 0.9 cells/1000 cells/hour). Provided that this topological difference in proliferation within the explant is recognised the in vitro model, in particular the basal level found at the centre, may still be regarded as a valid system for studying tissue responses to carcinogens and trophic factors.

Using a system designed to preserve, in vitro, both the epithelial and the connective tissue elements, we have maintained adult colonic mucosa in organ culture for up to 336 hours and have investigated the sequential morphological changes which occur. During the first 48 hours, normal micro-architecture is preserved, but there is progressive loss of cytoplasmic mucin from crypt cells. Subsequently, accelerated degenerative changes develop; cells are lost from the crypts and, because cell proliferation in the crypt is reduced, these lost cells are not replaced. For a time, the crypts are represented by discrete acinar formations or clusters of cells in the lamina propria, apparently discontinuous with the intact surface epithelial layer. These remnants manifest intense proliferative activity during the period between 72 and 96 hours after explantation, leading to the restoration of well formed crypts lined by columnar epithelial cells between 120 and 144 hours; differentiation of goblet cells ensues and this state of virtually normal structure persists until the termination of culture between 186 and 336 hours. It is concluded that the determination of normal crypt structure and of crypt-cell differentiation is governed by intrinsic control mechanisms although these may be subject to extrinsic modulation. Whether or not the degeneration phase can be eliminated, it is clear that long term culture of adult human colonic mucosa is possible. Such a system may be useful in the study of mucosal function and of mucosal response to drugs, carcinogens and trophic factors.