The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis

Although necrosis and necroinflammation are central features of many liver diseases, the role of programmed necrosis in the context of inflammation-dependent hepatocellular death remains to be fully determined. Here, we have demonstrated that the pseudokinase mixed lineage kinase domain-like protein (MLKL), which plays a key role in the execution of receptor interacting protein (RIP) lcinase-dependent necroptosis, is upregulated and activated in human autoimmune hepatitis and in a murine model of inflammation-dependent hepatitis. Using genetic and pharmacologic approaches, we determined that hepatocellular necrosis in experimental hepatitis is driven by an MLKL-dependent pathway that occurs independently of RIPK3. Moreover, we have provided evidence that the cytotoxic activity of the proinflammatory cytokine IFN-gamma in hepatic inflammation is strongly connected to induction of MLKL expression via activation of the transcription factor STAT1. In summary, our results reveal a pathway for MLKL-dependent programmed necrosis that is executed in the absence of RIPK3 and potentially drives the pathogenesis of severe liver diseases

Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

Acknowledgements We thank the Red Cross blood bank in Melbourne for human erythrocytes. We thank Svenja Gunther for expression of GBP130 66–196 proteins; Michelle Gazdik and Chris Burns for help in preparing lipids; Lachlan Whitehead (Centre for Dynamic Imaging, Walter and Eliza Hall Institute) for assistance with quantification of export; and David Bocher for help with generation of STEVOR constructs. This work was supported by the National Health and Medical Research Council of Australia (grants 637406, 1010326, 1049811 and 1057960), a Ramaciotti Foundation Establishment Grant (3197/2010), a Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS, and the Canadian Institutes for Health Research (MOP#130359). J.A.B is an Australian Research Council QEII Fellow, SF was supported by the Research Training Group GRK1459 of the German Research Foundation, and AFC is a Howard Hughes International Scholar.Peer reviewedPublisher PD

REDD+, transformational change and the promise of performance-based payments: a qualitative comparative analysis

Reducing emissions from deforestation and forest degradation (REDD+) has emerged as a promising climate change mitigation mechanism in developing countries. This paper examines the national political context in 13 REDD+ countries in order to identify the enabling conditions for achieving progress with the implementation of countries REDD+ policies and measures. The analysis builds on a qualitative comparative analysis (QCA) of various countries' progress with REDD+, conducted in 12 REDD+ countries in 2012, which highlighted the importance of factors such as already initiated policy change, and the presence of coalitions calling for broader policy change A follow-up survey in 2014 was considered timely because the REDD+ policy arena, at international and at country levels, is highly dynamic and undergoes constant evolution, which affects progress with REDD+ policy making and implementation. Furthermore, we will now examine whether the 'promise' of performance-based funds has played a role in enabling the establishment of REDD+. The results show a set of enabling conditions and characteristics of the policy process under which REDD+ policies can be established. The study finds that the existence of broader policy change, and availability of performance-based funding in combination with strong national ownership of the REDD+ policy process may help guide other countries seeking to formulate REDD+ policies that are likely to deliver efficient, effective, and equitable outcomes

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains

Chemical cleavage reactions of DNA on solid support: application in mutation detection

BACKGROUND: The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. RESULTS: DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO(4 )(potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. CONCLUSIONS: The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations

Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells.

The spontaneously immortalised DF-1 cell line is rapidly replacing its progenitor primary chicken embryo fibroblasts (CEFs) for studies on avian viruses such as avian influenza but no comprehensive study has as yet been reported comparing their innate immunity phenotypes. We conducted microarray analyses of DF-1 and CEFs, under both normal and stimulated conditions using chicken interferon-α (chIFN-α) and the attenuated infectious bursal disease virus vaccine strain PBG98. We found that DF-1 have an attenuated innate response compared to CEFs. Basal expression levels of Suppressor of Cytokine Signalling 1 (chSOCS1), a negative regulator of cytokine signalling in mammals, are 16-fold higher in DF-1 than in CEFs. The chSOCS1 “SOCS box” domain (which in mammals, interacts with an E3 ubiquitin ligase complex) is not essential for the inhibition of cytokine-induced JAK/STAT signalling activation in DF-1. Overexpression of SOCS1 in chIFN-α-stimulated DF-1 led to a relative decrease in expression of interferon-stimulated genes (ISGs; MX1 and IFIT5) and increased viral yield in response to PBG98 infection. Conversely, knockdown of SOCS1 enhanced induction of ISGs and reduced viral yield in chIFN-α-stimulated DF-1. Consequently, SOCS1 reduces induction of the IFN signalling pathway in chicken cells and can potentiate virus replication