New Caledonia harbours a highly diverse and endemic flora, and 13 (out of the 19 worldwide) species of Araucaria are endemic to this territory. Their phylogenetic relationships remain largely unresolved. Using nuclear microsatellites and chloroplast DNA sequencing, we focused on five closely related Araucaria species to investigate among-species relationships and the distribution of within-species genetic diversity across New Caledonia.

GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3(S408A,S409A)γ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3(S408A,S409A)δ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4(S443A)β3(S408A,S409A)δ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3(S408,S409) implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by two endogenous neuromodulators.

Alcohol dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) β1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN x C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse.

Purpose: To explore in a small pilot study whether oral melatonin, administered during ovarian stimulation increases clinical pregnancy rate (CPR) after IVF and what dose might be most effective. Methods: Pilot double-blind, dose-finding, placebo-controlled randomized clinical trial in private IVF clinics in Australia between September 2014 and September 2016. One hundred and sixty women having their first cycle of IVF or ICSI were randomized to receive placebo (n = 40), melatonin 2 mg (n = 41), melatonin 4 mg (n = 39), or melatonin 8 mg (n = 40) twice per day (BD) during ovarian stimulation. The primary outcome was CPR. Secondary outcomes included serum and follicular fluid (FF) melatonin concentrations, oocyte/embryo quantity/quality, and live birth rate (LBR). Analysis was performed using the intention-to-treat principle. Results: There was no difference in CPR or LBR between any of the four groups (p = 0.5). When all the doses of melatonin were compared as a group with placebo, the CPR was 21.7% for the former and 15.0% for the latter [OR 1.57 (95% CI 0.59, 4.14), p = 0.4]. There were also no differences between the groups in total oocyte number, number of MII oocytes, number of fertilized oocytes, or the number or quality of embryos between the groups. This is despite mean FF melatonin concentration in the highest dose group (8 mg BD) being nine-fold higher compared with placebo (P < 0.001). Conclusion: No significant differences were observed in CPR or oocyte and embryo parameters despite finding a nine-fold increase in FF melatonin concentration. However, this study was not sufficiently powered to assess differences in CPR and therefore, these results should be interpreted with caution. Because this was a small RCT, a beneficial effect of melatonin on IVF pregnancy rates cannot be excluded and merits confirmation in further, larger clinical trials. ANZCTR (http://www.anzctr.org.au/ Project ID: ACTRN12613001317785).

GABAA receptors (GABAARs) are the principal inhibitory neurotransmitter receptors in the central nervous system. They control neuronal excitability by synaptic and tonic forms of inhibition mostly mediated by different receptor subtypes located in specific cell membrane subdomains. A consensus suggests that α1-3βγ comprise synaptic GABAARs, whilst extrasynaptic α4βδ, α5βγ and αβ isoforms largely underlie tonic inhibition. Although some structural features that enable the spatial segregation of receptors are known, the mobility of key synaptic and extrasynaptic GABAARs are less understood, and yet this is a key determinant of the efficacy of GABA inhibition. To address this aspect, we have incorporated functionally silent α-bungarotoxin binding sites (BBS) into prominent hippocampal GABAAR subunits which mediate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABAARs that are traditionally considered to mediate synaptic or tonic inhibition are all able to access inhibitory synapses. These isoforms have variable diffusion rates and are differentially retained upon entering the synaptic membrane subdomain. Interestingly, α2 and α4 subunits reside longer at synapses compared to α5 and δ subunits. Furthermore, a high proportion of extrasynaptic δ-containing receptors exhibited slower diffusion compared to δ subunits at synapses. A chimera formed from δ-subunits, with the intracellular domain of γ2L, reversed this behaviour. In addition, we observed that receptor activation affected the diffusion of extrasynaptic, but not of synaptic GABAARs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABAARs are determined by receptor subunit composition and intracellular structural motifs. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

The question of what determines divergence both between and within species has been the central topic in evolutionary biology. Neutral drift and environmentally dependent divergence are predicted to play roles in driving population and lineage divergence. However, neutral drift may preclude adaptation if the rate of gene flow between populations is high. Here, we sampled populations of three Taiwania (Taiwania cryptomerioides) lineages occurring in Taiwan, the mainland of China (Yunnan-Myanmar border), and northern Vietnam, and tested the relative strength of neutral drift and divergent selection in shaping divergence of those populations and lineages. We quantified genetic and epigenetic variation, respectively, using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). Analysis of 1413 AFLP and 462 MSAP loci using frequency-based genome scan methods and generalized linear models (GLMs) found no potential selective outliers when only Taiwanese populations were examined, suggesting that neutral drift was the predominant evolutionary process driving differentiation between those populations. However, environmentally associated divergence was found when lineages were compared. Thirty-two potential selective outliers were identified based on genome scans and their associations with environmental variables were tested with GLMs, generalized linear mixed effect models (GLMMs), and model selection with a model averaging approach. Ten loci (six AFLP and four MSAP) were found to be strongly associated with environmental variables, particularly monthly temperature variation and normalized difference vegetation index (NDVI) using model selection and a model averaging approach. Because only a small portion of genetic and epigenetic loci were found to be potential selective outliers, neutral evolutionary process might also have played crucial roles in driving lineage divergence, particularly between geographically and genetically isolated island and mainland Asia lineages. Nevertheless, the vast amount of neutral drift causing genetic and epigenetic variations might have the potential for adaptation to future climate changes. These could be important for the survival of Taiwania in different geographic areas.

Glutathione has a wide range of functions; it is an endogenous anti-oxidant and plays a key role in the maintenance of intracellular redox balance and detoxification of xenobiotics. Several studies have indicated that children with autism spectrum disorders may have altered glutathione metabolism which could play a key role in the condition.

Aggresomes are pericentrosomal accumulations of misfolded proteins, chaperones and proteasomes. Their positioning near the centrosome, like that of other organelles, requires active, microtubule-dependent transport. Linker proteins that can associate with the motor protein dynein, organelles, and microtubules are thought to contribute to the active maintenance of the juxtanuclear localization of many membrane bound organelles and aggresomes. Hook proteins have been proposed to serve as adaptors for the association of cargos with dynein for transport on microtubules. Hook2 was shown to localize to the centrosome, bind centriolin, and contribute to centrosomal function.

The ubiquitous ribosome-associated complex (RAC) is a chaperone that spans ribosomes, making contacts near both the polypeptide exit tunnel and the decoding center, a position prime for sensing and coordinating translation and folding. Loss of RAC is known to result in growth defects and sensitization to translational and osmotic stresses. However, the physiological substrates of RAC and the mechanism(s) by which RAC is involved in responding to specific stresses in higher eukaryotes remain obscure. The data presented here uncover an essential function of mammalian RAC in the unfolded protein response (UPR). Knockdown of RAC sensitizes mammalian cells to endoplasmic reticulum (ER) stress and selectively interferes with IRE1 branch activation. Higher-order oligomerization of the inositol-requiring enzyme 1α (IRE1α) kinase/endoribonuclease depends upon RAC. These results reveal a surveillance function for RAC in the UPR, as follows: modulating IRE1α clustering as required for endonuclease activation and splicing of the substrate Xbp1 mRNA.

The plant hormone auxin is a conserved regulator of development which has been implicated in the generation of morphological novelty. PIN-FORMED1 (PIN) auxin efflux carriers are central to auxin function by regulating its distribution. PIN family members have divergent structures and cellular localizations, but the origin and evolutionary significance of this variation is unresolved. To characterize PIN family evolution, we have undertaken phylogenetic and structural analyses with a massive increase in taxon sampling over previous studies. Our phylogeny shows that following the divergence of the bryophyte and lycophyte lineages, two deep duplication events gave rise to three distinct lineages of PIN proteins in euphyllophytes. Subsequent independent radiations within each of these lineages were taxonomically asymmetric, giving rise to at least 21 clades of PIN proteins, of which 15 are revealed here for the first time. Although most PIN protein clades share a conserved canonical structure with a modular central loop domain, a small number of noncanonical clades dispersed across the phylogeny have highly divergent protein structure. We propose that PIN proteins underwent sub- and neofunctionalization with substantial modification to protein structure throughout plant evolution. Our results have important implications for plant evolution as they suggest that structurally divergent PIN proteins that arose in paralogous radiations contributed to the convergent evolution of organ systems in different land plant lineages.

The relationship between sleep and micronutrients, including magnesium, is implicated in its regulation. The effects of low magnesium and other micronutrients on sleep disruption and telomere loss are not well understood. The present study was carried out in 172 healthy elderly subjects from South Australia. Plasma micronutrients including magnesium were measured. Each participant provided information about their sleep hours (<7 h or ≥7 h). Lymphocyte telomere length (TL) was measured by real-time qPCR assay. Plasma magnesium level was significantly low in subjects who sleep less than 7 h (p = 0.0002). TL was significantly shorter in people who are low in magnesium and sleep less than 7 h (p = 0.01). Plasma homocysteine (Hcy) is negatively associated with magnesium (r = −0.299; p < 0.0001). There is a significant interaction effect of magnesium and Hcy on sleep duration (p = 0.04) and TL (p = 0.003). Our results suggest that inadequate magnesium levels have an adverse impact on sleep and telomere attrition rate in cognitively normal elderly people, and this may be exacerbated by low levels of vitamin B12 and folate that elevate Hcy concentration.

New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a 'museum' for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.

Despite the availability of several studies to clarify taxonomic problems on the highly threatened yews of the Hindu Kush-Himalaya (HKH) and adjacent regions, the total number of species and their exact distribution ranges remains controversial. We explored the use of comprehensive sets of morphological, molecular and climatic data to clarify taxonomy and distributions of yews in this region.

Driven by limited resources and a sense of urgency, the prioritization of species for conservation has been a persistent concern in conservation science. Gymnosperms (comprising ginkgo, conifers, cycads, and gnetophytes) are one of the most threatened groups of living organisms, with 40% of the species at high risk of extinction, about twice as many as the most recent estimates for all plants (i.e. 21.4%). This high proportion of species facing extinction highlights the urgent action required to secure their future through an objective prioritization approach. The Evolutionary Distinct and Globally Endangered (EDGE) method rapidly ranks species based on their evolutionary distinctiveness and the extinction risks they face. EDGE is applied to gymnosperms using a phylogenetic tree comprising DNA sequence data for 85% of gymnosperm species (923 out of 1090 species), to which the 167 missing species were added, and IUCN Red List assessments available for 92% of species. The effect of different extinction probability transformations and the handling of IUCN data deficient species on the resulting rankings is investigated. Although top entries in our ranking comprise species that were expected to score well (e.g. Wollemia nobilis, Ginkgo biloba), many were unexpected (e.g. Araucaria araucana). These results highlight the necessity of using approaches that integrate evolutionary information in conservation science.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs.

Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABA(A) receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABA(A) receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABA(A) receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABA(A) receptors and is abolished by preventing CaMKII phosphorylation of GABA(A) receptors. Our results reveal a novel GABA(A) receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.