Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds

We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains’ expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our ‘hits’ have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness.     

Natural climate solutions versus bioenergy: Can carbon benefits of natural succession compete with bioenergy from short rotation coppice?

Short rotation plantations are often considered as holding vast potentials for future global bioenergy supply. In contrast to raising biomass harvests in forests, purpose‐grown biomass does not interfere with forest carbon (C) stocks. Provided that agricultural land can be diverted from food and feed production without impairing food security, energy plantations on current agricultural land appear as a beneficial option in terms of renewable, climate‐friendly energy supply. However, instead of supporting energy plantations, land could also be devoted to natural succession. It then acts as a long‐term C sink which also results in C benefits. We here compare the sink strength of natural succession on arable land with the C saving effects of bioenergy from plantations. Using geographically explicit data on global cropland distribution among climate and ecological zones, regionally specific C accumulation rates are calculated with IPCC default methods and values. C savings from bioenergy are given for a range of displacement factors (DFs), acknowledging the varying efficiency of bioenergy routes and technologies in fossil fuel displacement. A uniform spatial pattern is assumed for succession and bioenergy plantations, and the considered timeframes range from 20 to 100 years. For many parameter settings—in particular, longer timeframes and high DFs—bioenergy yields higher cumulative C savings than natural succession. Still, if woody biomass displaces liquid transport fuels or natural gas‐based electricity generation, natural succession is competitive or even superior for timeframes of 20–50 years. This finding has strong implications with climate and environmental policies: Freeing land for natural succession is a worthwhile low‐cost natural climate solution that has many co‐benefits for biodiversity and other ecosystem services. A considerable risk, however, is C stock losses (i.e., emissions) due to disturbances or land conversion at a later time.     

Comparison of T Cell Receptor-Induced Proximal Signaling and Downstream Functions in Immortalized and Primary T Cells

Background

Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies using human T cell lines have provided substantial insight into these signaling pathways, no comprehensive, direct comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness as a model of primary T cells.

Methodology/Principal Findings

We used quantitative biochemical techniques to compare the activation of two widely used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in proximal TCR-mediated signaling events. In contrast, Jurkat E6.1 cells had significantly increased site-specific phosphorylation of Pyk2, PLCγ1, Vav1, and Erk1/Erk2 and substantially more Ca²⁺ flux compared to HuT78 cells and APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of cytokines and chemokines released upon TCR and costimulatory receptor activation.

Conclusions/Significance

Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs. Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T cell line.     

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope‐coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N‐termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS‐PAGE, in‐gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI‐TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of ¹²C and ¹³C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.     

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation

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Structure and Dynamics of a Compact State of a Multidomain Protein,the Mercuric Ion Reductase

The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.     

Males of a single-brooded tropical bird species do not show increases in testosterone during social challenges

The challenge hypothesis poses that in socially monogamous vertebrates, males increase circulating testosterone in response to aggressive challenges to promote intense and persistent aggression. However, in bird species that raise only a single brood during short breeding seasons as well as those with essential male parental care, males lack the well-documented testosterone response to social challenges. We tested male behavioral and hormonal responses to social challenges in a neotropical bird species, the buff-breasted wren (Thryothorus leucotis), which is single-brooded with extensive male parental care, but in contrast to most species studied to date, has a long breeding season. We presented live female, male, and paired decoys with song playback for 30 min during pre-breeding and breeding periods. Males responded aggressively to all intruders, but male decoys elicited somewhat weaker responses overall. Responses to female decoys were most intense during pre-breeding, whereas pair decoys elicited stronger responses at breeding. Plasma testosterone concentrations did not differ between challenged and unchallenged males, or among males exposed to different decoys or during different seasons. Plasma corticosterone in pre-breeding males was higher in challenged than unchallenged males and varied positively with the duration of social challenge. Circulating dehydroepiandrosterone concentrations were similar in challenged and unchallenged males, but correlated positively with the proportion of time males spent in close proximity to the decoy. Both testosterone and corticosterone results support recent findings, suggesting that brood number and essential male care, but not breeding-season length, may be important determinants of male hormonal responsiveness during aggressive interactions.     

Role for plasmacytoid dendritic cells in anti-HIV innate immunity

The role of plasmacytoid dendritic cells (pDC) in anti-HIV immunity is mostly represented by the production of type I IFN in response to HIV infection in vitro and in vivo. This production is decreased in HIV-1 infected patients at the time of primary infection and during chronic disease in association with progression of disease. Circulating pDC counts are decreased concomitantly with type I IFN, and both factors correlate inversely overall with viral loads and positively with CD4+ T-cell counts. These parameters might be used in clinical immunology to monitor treatment and as predictive factors of immune control of HIV-1 replication to help decide whether to interrupt antiretroviral treatment. They may be related to control of HIV replication as well as to pathogenesis of infection, perhaps in setting the balance between immunity or tolerance to the virus. A better understanding of these parameters is required while attempts to use IFN-alpha or ligands of Toll-like receptors found on pDC are being made.