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The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel’s role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G-protein coupled receptors (GPCR) that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates GPCRs and will aid in elucidating the pathways involved in metabolic dysfunction.

Scavenging birds can provide ecosystem services to pastoralists by contributing to the breakdown of animal carcasses that can harbour and spread disease. However, these benefits have yet to be quantified in Australia. We monitored rabbit carcasses using motion-sensor cameras to identify beneficial avian scavengers across four landscape types (forest, riparian, fields with large isolated trees, and open fields) on a pastoral property in north-central Victoria. We quantified the ecosystem service of carcass breakdown by measuring the per cent weight loss of carcasses exposed to scavenging birds compared to carcasses excluded from birds. Seven of the twenty-four bird-accessible carcasses were attacked by raptors in 2014, and three in 2015. When a raptor attacked a carcass, there was a significantly higher median per cent weight loss of the carcass (16.67%, interquartile range (IQR) = 8.33–100.0) compared with sites where no bird attacks occurred (6.65%, IQR = 3.03–12.06). Our results indicate that ra...

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl β-d-glucoside and methyl 6-thio-β-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-β-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases., Enzyme substrates and products often diffuse too rapidly to assess the catalytic implications of these movements. Here, the authors characterise the structural basis of product and substrate diffusion for an exo-hydrolase and discover a substrate-product assisted processive catalytic mechanism.

Prospective memory is remembering to carry out a behavior on a particular occasion or at a specific time in the future. This form of remembering is critical for the daily functioning of children with autism spectrum disorder (ASD) and their functional independence from caregivers. We used a single-subject design to investigate whether reinforcement increased the accuracy of prospective remembering in the context of a computerized board game, Virtual Week, of four 6- to 7-year-old children diagnosed with ASD. Reinforcement increased accuracy for all participants compared to baseline performance and effects were maintained after reinforcement was discontinued for three of four children. This is the first study of which we are aware to use a reinforcement-based behavioral intervention to improve the prospective remembering of children with ASD. Limitations and areas for future research are discussed.

Birds active in apple orchards in south–eastern Australia can contribute positively (e.g., control crop pests) or negatively (e.g., crop damage) to crop yields. Our study is the first to identify net outcomes of these activities, using six apple orchards, varying in management intensity, in south–eastern Australia as a study system. We also conducted a predation experiment using real and artificial codling moth (Cydia pomonella) larvae (a major pest in apple crops). We found that: (1) excluding birds from branches of apple trees resulted in an average of 12.8% more apples damaged by insects; (2) bird damage to apples was low (1.9% of apples); and (3) when trading off the potential benefits (biological control) with costs (bird damage to apples), birds provided an overall net benefit to orchard growers. We found that predation of real codling moth larvae was higher than for plasticine larvae, suggesting that plasticine prey models are not useful for inferring actual predation levels. Our study shows how complex ecological interactions between birds and invertebrates affect crop yield in apples, and provides practical strategies for improving the sustainability of orchard systems.

Skilled and knowledgeable staff equipped to respond to change is a key learning and growth goal of the University of Wollongong Library. But how do front line staff develop and maintain their client-centred service skills and behaviours? How do they manage the myriad of diverse needs and expectations from a constantly changing student population, within a volatile technological environment? By challenging established approaches to service delivery training, it was possible to demonstrate a need to be more adaptable and responsive. We recognized that earlier approaches such as on-the-job training were no longer adequate. In 2009 the University of Wollongong Library introduced competency-based training. This represented a new approach to staff training and an opportunity for renewal. Numerous factors contributed to this decision, with the overarching goal being to keep our staff on the cutting edge of service delivery. A systematic approach was used that involved researching competency-based trainin...

MDA5, an RIG-I-like helicase, is a conserved cytoplasmic viral RNA sensor, which recognizes dsRNA from a wide-range of viruses in a length-dependent manner. It has been proposed that MDA5 forms higher-order structures upon viral dsRNA recognition or during antiviral signaling, however the organization and nature of this proposed oligomeric state is unknown. We report here that MDA5 cooperatively assembles into a filamentous oligomer composed of a repeating segmental arrangement of MDA5 dimers along the length of dsRNA. Binding of MDA5 to dsRNA stimulates its ATP hydrolysis activity with little coordination between neighboring molecules within a filament. Individual ATP hydrolysis in turn renders an intrinsic kinetic instability to the MDA5 filament, triggering dissociation of MDA5 from dsRNA at a rate inversely proportional to the filament length. These results suggest a previously unrecognized role of ATP hydrolysis in control of filament assembly and disassembly processes, thereby autoregulating the interaction of MDA5 with dsRNA, and provides a potential basis for dsRNA length-dependent antiviral signaling.

We report a method for the expression in Escherichia coli of the isolated second type II fibronectin domain from MMP-2 (FNII-2). FNII-2 was expressed as a His(6)thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation. When cultured at 37 degrees C, the expressed protein is located exclusively in the soluble fraction of the E. coli lysate. The fusion protein from the soluble fraction was purified and the His(6)thioredoxin-tag was cleaved by thrombin, resulting in a yield of approximately 40 mg/L. The recombinant FNII-2 was demonstrated to be functional by its ability to bind to gelatin-Sepharose, correct folding of the purified protein was confirmed by NMR spectroscopy. This approach may generally be applicable to all FNII domains and is a significant simplification relative to existing techniques involving refolding from inclusion bodies or expression in the eukaryotic host, Pichia pastoris.

While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (150 kDa) complexes between GPCRs and their partner proteins. Negative stain EM, whereby the biological samples are embedded in a thin layer of heavy metal solution, is a well-established alternative technique that provides the enhanced contrast needed to visualize small macromolecular complexes. This methodology can offer a simple and powerful tool for the rapid evaluation of sample characteristics, such as homogeneity or oligomeric state. When coupled to single particle classification and averaging, negative stain EM can provide valuable information on the overall architecture and dynamics of protein complexes. Here we provide a concise protocol for negative stain imaging and two-dimensional (2D) projection analysis of GPCR complexes, including notes for the intricacies of the application in these biological systems.

While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (

SUMMARY The vertebrate antiviral innate immune system is often considered to consist of two distinct groups of proteins: pattern recognition receptors (PRRs) that detect viral infection and induce the interferon (IFN) signaling, and effectors that directly act against viral replication. Accordingly, previous studies on PRRs, such as RIG-I and MDA5, have primarily focused on their functions in viral double-stranded RNA (dsRNA) detection and consequent antiviral signaling. We report here that both RIG-I and MDA5 efficiently displace viral proteins pre-bound to dsRNA in a manner dependent on their ATP hydrolysis, and that this activity assists a dsRNAdependent antiviral effector protein, PKR, and allows RIG-I to promote MDA5 signaling. Furthermore, truncated RIG-I/MDA5 lacking the signaling domain, and hence the IFN stimulatory activity, displaces viral proteins and suppresses replication of certain viruses in an ATP-dependent manner. Thus, this study reveals novel ‘‘effector-like’’ functions of RIG-I and MDA5 that challenge the conventional view of PRRs.