Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen

<b>Background</b><br /> The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.<p></p> <b>Results</b><br /> Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.<p></p> <b>Conclusion</b><br /> Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.<p></p&gt

Report on a visit to Sri Lanka to study technical and socio-economic aspects of the BOBP Post-harvest Fisheries Programme

The team consider that the general marketing and postharvest handling of fish and fish products in Sri Lanka is efficient given the resource constraints. Conditions of under supply and high consumer demand promote competition. The fisheries sector is highly dynamic and responsive to changes in demand. Post-harvest losses in value, and quantity are minimal, though some issues of quality were identified

Selection Among and Within S1 Lines of Maize on S2 Line and Testcross Performance

Most maize (Zea mays L.) breeders practice visual selection among lines during inbreeding, but may not be certain of the effectiveness of such selection. Visual selection among and within 1,636 S1 lines of maize derived from \u27Lancaster Composite\u27 was used to select 200 S2 lines, and a random set of 200 S2 lines also was developed. Yield trials of the 400 S2 lines in three environments and their testcrosses to (B73 x B84) in four environments were conducted to determine whether visual selection was effective in choosing high-yielding and agronomically desirable lines with superior combining ability. Grain yield of the visually selected S1 lines (3.11 Mg ha-1) was significantly (P\u3c0.05) greater than that of the unselected lines (2.94 Mg ha-1), but there was no difference in testcross means. Visually selected S1 lines had slightly greater mean grain moisture and slightly less mean stalk lodging than unselected lines in individual environments. Testcrosses of visually selected lines had greater grain moisture and less stalk lodging than testcrosses of unselected lines in individual environments. Estimates of genetic variance, heritability, and gain from selection were not consistently affected by visual selection. Many superior S2 lines and testcrosses were unselected lines, showing that visual selection failed to identify many desirable genotypes. Our results suggest that visual selection should not be used to attempt to select the most superior genotypes, but should emphasize discarding of undesirable genotypes before yield testing

Fabrication of a compliant phantom of the human aortic arch for use in Particle Image Velocimetry (PIV) experimentation

Compliant phantoms of the human aortic arch can mimic patient specific cardiovascular dysfunctions in vitro. Hence, phantoms may enable elucidation of haemodynamic disturbances caused by aortic dysfunction. This paper describes the fabrication of a thin-walled silicone phantom of the human ascending aorta and brachiocephalic artery. The model geometry was determined via a meta-analysis and modelled in SolidWorks before 3D printing. The solid model surface was smoothed and scanned with a 3D scanner. An offset outer mould was milled from Ebalta S-Model board. The final phantom indicated that ABS was a suitable material for the internal model, the Ebalta S-Model board yielded a rough external surface. Co-location of the moulds during silicone pour was insufficient to enable consistent wall thickness. The resulting phantom was free of air bubbles but did not have the desired wall thickness consistency

Fate specification and tissue-specific cell cycle control of the <i>Caenorhabditis elegans</i> intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Evaluation of the International Child Development Programme (ICDP) as a community-wide parenting programme

Background: Many parenting programmes lack proper evaluation, especially under community-wide implementation. Objective: Examining the effectiveness of the eight-week International Child Development Programme (ICDP), implemented as a general programme. Methodology: Non-clinical caregivers attending ICDP (N = 141) and a non-attending community comparison group (N = 79) completed questionnaires on parenting, psychosocial functioning, and child difficulties before and after ICDP course. Analyses compare changes in scores for both groups over time. Results: The ICDP group showed more positive attitudes towards child management and reported better child management, improved parental strategies and less impact of child difficulties. Caregivers with low initial scores benefited most. The comparison group showed little change with a significant decrease in scores on the caregiver–child activity scale. Discussion: The results suggest that caregivers in the community who do not show clinical signs or have children with behaviour or other disorders, may benefit from participating in parent training based on ICDP

The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell divisio