Biodiversity on the brink: Evaluating a transdisciplinary research collaboration

Global biodiversity is facing an extinction crisis. Australia has one of the highest terrestrial species extinction rates in the world. Scientists, policy advisors and governments have recommended that the issue be addressed at a landscape-scale, while noting that there are significant knowledge gaps that are hampering implementation of such an approach. From 2011–2015, the Australian Government funded a transdisciplinary research program, the Landscapes and Policy Hub, to meet this need. Transdisciplinary research is widely acknowledged as essential to address the complexity of contemporary environmental problems. Given that such research programs are in their infancy, it is important to evaluate their efficacy and provide an empirical basis for improving their design. This paper presents an evaluation of the strategies fostering transdisciplinarity adopted by the Landscapes and Policy Hub. A heavy emphasis on communication, with skilled knowledge brokering, regular face-to-face meetings using participatory activities and shared field engagements enhanced transdisciplinary interaction between researchers and research users. However, establishing a fully integrated interdisciplinary research program remained a challenge. Efforts to enable shared conceptual frameworks to emerge through adaptive application of theory in practice could have been balanced with increased effort at the outset for researchers and research users to collaboratively formulate shared research questions, leading to the establishment of teams that could address these questions through cross-mobilisation of interdisciplinary expertise.     

Imaging emotional brain functions: Conceptual and methodological issues

This article reviews the psychophysiological and brain imaging literature on emotional brain function from a methodological point of view. The difficulties in defining, operationalising and measuring emotional activation and, in particular, aversive learning will be considered. Emotion is a response of the organism during an episode of major significance and involves physiological activation, motivational, perceptual, evaluative and learning processes, motor expression, action tendencies and monitoring/subjective feelings. Despite the advances in assessing the physiological correlates of emotional perception and learning processes, a critical appraisal shows that functional neuroimaging approaches encounter methodological difficulties regarding measurement precision (e.g., response scaling and reproducibility) and validity (e.g., response specificity, generalisation to other paradigms, subjects or settings). Since emotional processes are not only the result of localised but also of widely distributed activation, a more representative model of assessment is needed that systematically relates the hierarchy of high- and low-level emotion constructs with the corresponding patterns of activity and functional connectivity of the brain.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

Temperature effects of intraventricular serotonin,norepinephrine and pilocarpine in the morphine-tolerant rat

Studies were undertaken to examine the possibility that changes occur in the responsiveness of thermoregulatory neurons in the anterior hypothalamus to endogenously released neurotransmitters upon the development of tolerance to morphine. In experiments conducted at an environmental temperature of 20–21°C, tolerance produced by the subcutaneous implantation of morphine alkaloid pellets failed to alter the temperature response of rats to intraventricular injections of 5-HT (10 μg), NE (20 μg) or pilocarpine (100 μg). It is concluded that tolerance development to morphine-induced hypothermia is not a result of changes in the postsynaptic sensitivity to the putative neurotransmitters in the thermoregulatory center.     

Kinetic analysis of blood-brain barrier transport of amino acids

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of ¹⁴C-labelled amino acids and ³H₂O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15 s following injection. A brain uptake index (I_b) was determined from the ratio of ¹⁴C dpm in the brain tissue and the injection mixture divided by the same ratio for the ³H₂O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded K_m (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship $\text{P =}\text{V}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the I_b for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the ³H₂O reference 15 s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min^?1.     

Electrophysiological evidence for the autoregulation of β-cell secretion by insulin

Electrophysiological studies of cultured rat pancreatic β-cells using intracellular microelectrodes show that exogenous insulin over the range of 0.1–10.0 μg/ml inhibits the electrical activity due to 27.8 mM glucose in a dose-related manner. This inhibitory effect is manifested by a mean increase of the membrane potential from about ?20 to ?30 mV and inhibition of the manner of cells impaled showing spike activity from 60 to less than 10%. The inhibitory influence of insulin is rapid occuring within 5 min for the highest level used. The results provide evidence for a negative feedback role of insulin in regulating its own release.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

The structure of 180° supernumerary limbs and a hypothesis of their formation

The results of a detailed analysis of 100 supernumerary limbs generated by 180° ipsilateral rotation (on the same limb stump) of regeneration blastemas is presented. The limbs were analyzed in terms of their position of origin, frequency, cartilage structure by Victoria blue staining, and muscle structure by serial sections. Single, double, or triple supernumeraries can be produced at no unique position of origin, although the posterodorsal quadrant was preferred. Four classes of supernumerary limbs were generated by such operations—normal; double dorsal or double ventral; part normal/part mirror imaged; part normal/part inverted in approximately equal frequencies. After amputation of these supernumeraries the same muscle patterns are faithfully regenerated. A hypothesis to explain the production of these abnormal limbs is proposed based on the observed phenomenon of fusion of supernumerary blastemata, but their regenerative behaviour presents problems for current models of pattern formation. Similar results have been obtained with developing limb buds and the relation between development and regeneration is discussed.