Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase

Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ⁹cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ⁶-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ⁶ desaturase to Δ⁹ regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ⁶ to Δ⁹ selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ⁹ to Δ⁶ selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ⁶ position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Predictions regarding the mechanism of Δ⁶ regioselectivity of the Thunbergia alata desaturase based on X-ray crystallography and molecular dynamics simulations are confirmed by experiment.     

Genetic Basis of a Cognitive Complexity Metric

Relational complexity (RC) is a metric reflecting capacity limitation in relational processing. It plays a crucial role in higher cognitive processes and is an endophenotype for several disorders. However, the genetic underpinnings of complex relational processing have not been investigated. Using the classical twin model, we estimated the heritability of RC and genetic overlap with intelligence (IQ), reasoning, and working memory in a twin and sibling sample aged 15-29 years (N = 787). Further, in an exploratory search for genetic loci contributing to RC, we examined associated genetic markers and genes in our Discovery sample and selected loci for replication in four independent samples (ALSPAC, LBC1936, NTR, NCNG), followed by meta-analysis (N>6500) at the single marker level. Twin modelling showed RC is highly heritable (67%), has considerable genetic overlap with IQ (59%), and is a major component of genetic covariation between reasoning and working memory (72%). At the molecular level, we found preliminary support for four single-marker loci (one in the gene DGKB), and at a gene-based level for the NPS gene, having influence on cognition. These results indicate that genetic sources influencing relational processing are a key component of the genetic architecture of broader cognitive abilities. Further, they suggest a genetic cascade, whereby genetic factors influencing capacity limitation in relational processing have a flow-on effect to more complex cognitive traits, including reasoning and working memory, and ultimately, IQ.     

Molecular Relationships of New Guinean Three-Striped Dasyures, (Myoictis, Marsupialia: Dasyuridae)

Complete nucleotide sequences of the cytochrome b and 12S rRNA genes and partial sequences of the mitochondrial 16S rRNA gene and the nuclear ɛ-globin gene were obtained from multiple exemplars of the New Guinean dasyurid, Myoictis. Allozyme data were also obtained from most of the same animals. The molecular data show that the genus comprises a number of genetically distinct lineages which correspond with groups proposed by Woolley (2005) on the basis of a number of morphological traits, including the form of the tail i.e. Myoictis leucura (sp. nov.), M. melas, M. wallacei and M. wavicus (new status). Divergence dates estimated from the weighted-average distances for the combined cytochrome b and 12S rRNA data, calibrated with a dasyurid-thylacine divergence 25 million years ago, suggest that the early cladogenic events separating Myoictis took place in the late Miocene. Subsequent separation of M. wavicus and M. leucura from a common ancestor as well as some genetic differentiation within M. melas, took place in the medial Pliocene.     

The crystal structure of the human co-chaperone P58(IPK)

P58^IPK is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58^IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58^IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58^IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58^IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58^IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58^IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.     

Impacts of Rotational Grazing on Soil Carbon in Native Grass-Based Pastures in Southern Australia

Rotational grazing management strategies have been promoted as a way to improve the sustainability of native grass-based pasture systems. From disturbance ecology theory, rotational grazing relative to continuous grazing can increase pasture productivity by allowing vegetation to recover after short intense grazing periods. This project sought to assess whether soil organic carbon (SOC) stocks would also increase with adoption of rotational grazing management. Twelve pairs of rotationally and continuously grazed paddocks were sampled across a rainfall gradient in South Australia. Pasture productivity approximated as the normalized difference vegetation index (NDVI) was on average no different between management categories, but when the data from all sites were aggregated as log response ratios (rotational/continuous) a significant positive trend of increasing NDVI under rotational grazing relative to continuous grazing was found (R² = 0.52). Mean SOC stocks (0–30 cm) were 48.3 Mg C ha^-1 with a range of 20–80 Mg C ha^-1 across the study area with no differences between grazing management categories. SOC stocks were well correlated with rainfall and temperature (multiple linear regression R² = 0.61). After removing the influence of climate on SOC stocks, the management variables, rest periods, stocking rate and grazing days, were found to be significantly correlated with SOC, explaining 22% of the variance in SOC, but there were still no clear differences in SOC stocks at paired sites. We suggest three reasons for the lack of SOC response. First, changes in plant productivity and turnover in low-medium rainfall regions due to changes in grazing management are small and slow, so we would only expect at best small incremental changes in SOC stocks. This is compounded by the inherent variability within and between paddocks making detection of a small real change difficult on short timescales. Lastly, the management data suggests that there is a gradation in implementation of rotational grazing and the use of two fixed categories (i.e. rotational v. continuous) may not be the most appropriate method of comparing diverse management styles.     

Root Effect Haemoglobins in Fish May Greatly Enhance General Oxygen Delivery Relative to Other Vertebrates

The teleost fishes represent over half of all extant vertebrates; they occupy nearly every body of water and in doing so, occupy a diverse array of environmental conditions. We propose that their success is related to a unique oxygen (O₂) transport system involving their extremely pH-sensitive haemoglobin (Hb). A reduction in pH reduces both Hb-O₂ affinity (Bohr effect) and carrying capacity (Root effect). This, combined with a large arterial-venous pH change (ΔpH_a-v) relative to other vertebrates, may greatly enhance tissue oxygen delivery in teleosts (e.g., rainbow trout) during stress, beyond that in mammals (e.g., human). We generated oxygen equilibrium curves (OECs) at five different CO₂ tensions for rainbow trout and determined that, when Hb-O₂ saturation is 50% or greater, the change in oxygen partial pressure (ΔPO₂) associated with ΔpH_a-v can exceed that of the mammalian Bohr effect by at least 3-fold, but as much as 21-fold. Using known ΔpH_a-v and assuming a constant arterial-venous PO₂ difference (P_a-vO₂), Root effect Hbs can enhance O₂ release to the tissues by 73.5% in trout; whereas, the Bohr effect alone is responsible for enhancing O₂ release by only 1.3% in humans. Disequilibrium states are likely operational in teleosts in vivo, and therefore the ΔpH_a-v, and thus enhancement of O₂ delivery, could be even larger. Modeling with known P_a-vO₂ in fish during exercise and hypoxia indicates that O₂ release from the Hb and therefore potentially tissue O₂ delivery may double during exercise and triple during some levels of hypoxia. These characteristics may be central to performance of athletic fish species such as salmonids, but may indicate that general tissue oxygen delivery may have been the incipient function of Root effect Hbs in fish, a trait strongly associated with the adaptive radiation of teleosts.     

Population variation in the thermal response to climate change reveals differing sensitivity in a benthic shark

Many species with broad distributions are exposed to different thermal regimes which often select for varied phenotypes. This intraspecific variation is often overlooked but may be critical in dictating the vulnerability of different populations to environmental change. We reared Port Jackson shark (Heterodontus portusjacksoni) eggs from two thermally discrete populations (i.e. Jervis Bay and Adelaide) under each location's present‐day mean temperatures, predicted end‐of‐century temperatures and under reciprocal‐cross conditions to establish intraspecific thermal sensitivity. Rearing temperatures strongly influenced ?O_2Max and critical thermal limits, regardless of population, indicative of acclimation processes. However, there were significant population‐level effects, such that Jervis Bay sharks, regardless of rearing temperature, did not exhibit differences in ?O_2Rest, but under elevated temperatures exhibited reduced maximum swimming activity with step‐wise increases in temperature. In contrast, Adelaide sharks reared under elevated temperatures doubled their ?O_2Rest, relative to their present‐day temperature counterparts; however, maximum swimming activity was not influenced. With respect to reciprocal‐cross comparisons, few differences were detected between Jervis Bay and Adelaide sharks reared under ambient Jervis Bay temperatures. Similarly, juveniles (from both populations) reared under Adelaide conditions had similar thermal limits and swimming activity (maximum volitional velocity and distance) to each other, indicative of conserved acclimation capacity. However, under Adelaide temperatures, the ?O_2Rest of Jervis Bay sharks was greater than that of Adelaide sharks. This indicates that the energetics of cooler water population (Adelaide) is likely more thermally sensitive than that of the warmer population (Jervis Bay). While unique to elasmobranchs, these data provide further support that by treating species as static, homogeneous populations, we ignore the impacts of thermal history and intraspecific variation on thermal sensitivity. With climate change, intraspecific variation will manifest as populations move, demographics change or extirpations occur, starting with the most sensitive populations.     

Phase locking, the Moran effect and distance decay of synchrony: experimental tests in a model system

Spatially separated populations of many species fluctuate synchronously. Synchrony typically decays with increasing interpopulation distance. Spatial synchrony, and its distance decay, might reflect distance decay of environmental synchrony (the Moran effect), and/or short-distance dispersal. However, short-distance dispersal can synchronize entire metapopulations if within-patch dynamics are cyclic, a phenomenon known as phase locking. We manipulated the presence/absence of short-distance dispersal and spatially decaying environmental synchrony and examined their separate and interactive effects on the synchrony of the protist prey species Tetrahymena pyriformis growing in spatial arrays of patches (laboratory microcosms). The protist predator Euplotes patella consumed Tetrahymena and generated predator-prey cycles. Dispersal increased prey synchrony uniformly over both short and long distances, and did so by entraining the phases of the predator-prey cycles. The Moran effect also increased prey synchrony, but only over short distances where environmental synchrony was strongest, and did so by increasing the synchrony of stochastic fluctuations superimposed on the predator-prey cycle. Our results provide the first experimental demonstration of distance decay of synchrony due to distance decay of the Moran effect. Distance decay of the Moran effect likely explains distance decay of synchrony in many natural systems. Our results also provide an experimental demonstration of long-distance phase locking, and explain why cyclic populations provide many of the most dramatic examples of long-distance spatial synchrony in nature.