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. Nevertheless, triple null mutants (Δmca2/3Δmca5) could be isolated after sequential gene deletion. Thereafter, Δmca2/3Δmca5 mutants were found to grow well both in vitro in culture and in vivo in mice. We hypothesise that metacaspases are essential for bloodstream form parasites, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of Δmca2/3Δmca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin D2. The metacaspases were found to colocalise with RAB11, a marker for recycling endosomes. However, variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, Δmca2/3Δmca5 and triple RNAi induced parasites. Thus, the data provide no support for the direct involvement of T. brucei metacaspases in programmed cell death and suggest that the proteins have a function associated with RAB11 vesicles that is independent of known recycling processes of RAB11-positive endosomes

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light synchronised axenic Prochlorococcus (PCC9511 strain) culture and 35S-methionine and 3H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >104 times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G2 cell cycle stage were consistently 2.2 times higher than those of cells at the G1 stage. Furthermore, S+G2 cells up-regulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday, and up to 42% at dusk, of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations

Phosphorylation-dependent assembly of DNA damage response systems and the central roles of TOPBP1

The cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the post-translational modifications (PTM) of hundreds of proteins involved in the detection and repair of DNA damage, and the communication of the state of damage to the cellular systems that regulate replication and cell division. A substantial proportion of these PTMs involve targeted phosphorylation, which among other effects, promotes the formation of multiprotein complexes through the specific binding of phosphorylated motifs on one protein, by specialised domains on other proteins. Understanding the nature of these phosphorylation mediated interactions allows definition of the pathways and networks that coordinate the DDR, and helps identify new targets for therapeutic intervention that may be of benefit in the treatment of cancer, where DDR plays a key role. In this review we summarise the present understanding of how phosphorylated motifs are recognised by BRCT domains, which occur in many DDR proteins. We particularly focus on TOPBP1 – a multi-BRCT domain scaffold protein with essential roles in replication and the repair and signalling of DNA damage

Cryptic diversity found in Didymellaceae from Australian native legumes

Ascochyta koolunga (Didymellaceae, Pleosporales) was first described in 2009 (as Phoma koolunga) and identified as the causal agent of Ascochyta blight of Pisum sativum (field pea) in South Australia. Since then A. koolunga has not been reported anywhere else in the world, and its origins and occurrence on other legume (Fabaceae) species remains unknown. Blight and leaf spot diseases of Australian native, pasture and naturalised legumes were studied to investigate a possible native origin of A. koolunga. Ascochyta koolunga was not detected on native, naturalised or pasture legumes that had leaf spot symptoms, in any of the studied regions in southern Australia, and only one isolate was recovered from P. sativum. However, we isolated five novel species in the Didymellaceae from leaf spots of Australian native legumes from commercial field pea regions throughout southern Australia. The novel species were classified on the basis of morphology and phylogenetic analyses of the internal transcribed spacer region and part of the RNA polymerase II subunit B gene region. Three of these species, Nothophoma garlbiwalawarda sp. nov., Nothophoma naiawu sp. nov. and Nothophoma ngayawang sp. nov., were isolated from Senna artemisioides. The other species described here are Epicoccum djirangnandiri sp. nov. from Swainsona galegifolia and Neodidymelliopsis tinkyukuku sp. nov. from Hardenbergia violacea. In addition, we report three new host-pathogen associations in Australia, namely Didymella pinodes on S. artemisioides and Vicia cracca, and D. lethalis on Lathyrus tingitanus. This is also the first report of Didymella prosopidis in Australi

Draft genome sequence of isolate Staphylococcus aureus LHSKBClinical, isolated from an infected hip

We report here the genome sequence of a clinical isolate of <i>Staphylococcus aureus</i> from an orthopedic infection. Phenotypically diverse <i>Staphylococcus aureus</i> strains are associated with orthopedic infections and subsequent implant failure, and some are highly resistant to antibiotics. This genome sequence will support further analyses of strains causing orthopedic infections

Growing a Sustainable Biofuels Industry: Economics, Environmental Considerations, and the Role of the Conservation Reserve Program

Biofuels are expected to be a major contributor to renewable energy in the coming decades under the Renewable Fuel Standard (RFS). These fuels have many attractive properties including the promotion of energy independence, rural development, and the reduction of national carbon emissions. However, several unresolved environmental and economic concerns remain. Environmentally, much of the biomass is expected to come from agricultural expansion and/or intensification, which may greatly affect the net environmental impact, and economically, the lack of a developed infrastructure and bottlenecks along the supply chain may affect the industry\u27s economic vitality. The approximately 30 million acres (12 million hectares) under the Conservation Reserve Program (CRP) represent one land base for possible expansion. Here, we examine the potential role of the CRP in biofuels industry development, by (1) assessing the range of environmental effects on six end points of concern, and (2) simulating differences in potential industry growth nationally using a systems dynamics model. The model examines seven land-use scenarios (various percentages of CRP cultivation for biofuel) and five economic scenarios (subsidy schemes) to explore the benefits of using the CRP. The environmental assessment revealed wide variation in potential impacts. Lignocellulosic feedstocks had the greatest potential to improve the environmental condition relative to row crops, but the most plausible impacts were considered to be neutral or slightly negative. Model simulations revealed that industry growth was much more sensitive to economic scenarios than land-use scenarios—similar volumes of biofuels could be produced with no CRP as with 100% utilization. The range of responses to economic policy was substantial, including long-term market stagnation at current levels of first-generation biofuels under minimal policy intervention, or RFS-scale quantities of biofuels if policy or market conditions were more favorable. In total, the combination of the environmental assessment and the supply chain model suggests that large-scale conversion of the CRP to row crops would likely incur a significant environmental cost, without a concomitant benefit in terms of biofuel production

Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein

CHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN, a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR

Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4TopBP1

The BRCT-domain protein Rad4(TopBP1) facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3(ATR) -Rad26(ATRIP) kinase complex, and the Crb2(53BP1) mediator. We have now determined crystal structures of the BRCT repeats of Rad4(TopBP1), revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb2(53BP1). We identify a cluster of phosphorylation sites in the N-terminal region of Crb2(53BP1) that mediate interaction with Rad4(TopBP1) and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb2(53BP1) residues Thr215 and Thr235 promotes phosphorylation of the noncanonical Thr187 site by scaffolding cyclin-dependent kinase (CDK) recruitment. Finally, we show that the simultaneous interaction of a single Rad4(TopBP1) molecule with both Thr187 phosphorylation sites in a Crb2(53BP1) dimer is essential for establishing the DNA damage checkpoint

Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint

Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint