Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice

These preclinical studies aimed to 1) increase our understanding the dietary induction of nonalcoholic steatohepatitis (NASH), and, 2) further explore the utility and mechanisms of glucagon-like peptide-1 receptor (GLP-1R) agonism in NASH. We compared the effects of a high trans-fat (HTF) or high lard fat (HLF) diet on key facets of nonalcoholic fatty liver disease (NAFLD)/NASH in Lep(ob)/Lep(ob) and C57BL6J (B6) mice. Although HLF-fed mice experienced overall greater gains in weight and adiposity, the addition of trans-fat better mirrored pathophysiological features of NASH (e.g., hepatomegaly, hepatic lipid, and fibrosis). Administration of AC3174, an exenatide analog, and GLP-1R agonist to Lep(ob)/Lep(ob) and B6 ameliorated hepatic endpoints in both dietary models. Next, we assessed whether AC3174-mediated improvements in diet-induced NASH were solely due to weight loss in HTF-fed mice. AC3174-treatment significantly reduced body weight (8.3%), liver mass (14.2%), liver lipid (12.9%), plasma alanine aminotransferase, and triglycerides, whereas a calorie-restricted, weight-matched group demonstrated only modest nonsignificant reductions in liver mass (9%) and liver lipid (5.1%) relative to controls. Treatment of GLP-1R-deficient (GLP-1RKO) mice with AC3174 had no effect on body weight, adiposity, liver or plasma indices pointing to the GLP-1R-dependence of AC3174's effects. Interestingly, the role of endogenous GLP-1Rs in NASH merits further exploration as the GLP-1RKO model was protected from the deleterious hepatic effects of HTF. Our pharmacological data further support the clinical evaluation of the utility of GLP-1R agonists for treatment of NASH.     

Combined mitochondrial and nuclear data enhance resolution of a rapid radiation of Australian rainbow skinks (Scincidae: Carlia)

In contrast to low diversity seen in many Australian rainforest squamate genera, sclerophyll adapted groups—such as Carlia—show signs of faster diversification. Here we expand upon a previous single-locus mitochondrial DNA phylogenetic study of Carlia which described a major polytomy at an intermediate level of divergence. With additional mtDNA data, two nuclear intron loci and comprehensive taxonomic coverage, we provide support, congruent across loci, for the existence of three major clades. In doing so we recognise three genera for the ‘Carlia group of skinks’: clade 1, Carlia Gray, 1845; 2, Lygisaurus De Vis, 1884 (includes all species formerly known as Lygisaurus, and also includes C. parrhasius); 3, Liburnascincus Wells and Wellington, 1984 (consisting of three boulder-dwelling species). Remaining regions of low bootstrap and posterior probability support are associated with short internodes and apparent conflict among loci, as inferred by Partition Branch Support. Likelihood-based diversification-rate analysis rejects constant rate models, and indicates that Carlia underwent a period of relatively rapid diversification early in the evolution of the group, a rate 3–4 times faster than subsequent rates, and faster than comparable wet forest skinks.     

Characterisation of microsatellite loci in the subtidal habitat-forming alga, Phyllospora comosa (Phaeophyceae, Fucales)

Habitat-forming macroalgae play a central role in the ecology of temperate reefs worldwide but there exists a critical lack of knowledge about important processes such as dispersal and gene flow. Understanding dispersal and gene flow of habitat-forming seaweeds is particularly pertinent given that loss of habitat-forming algae is an increasingly prevalent problem worldwide. Here, we develop 10 microsatellite markers for the monotypic Phyllospora comosa, an important habitat-forming macroalga that has undergone massive declines on urbanised coastlines of Sydney, Australia. We characterise population genetic diversity and structure and estimate levels of dispersal and gene flow between the geographically isolated northern and southern populations in this subtidal macroalga.     

Where and when does a ring start and end? Testing the ring-species hypothesis in a species complex of Australian parrots

Speciation, despite ongoing gene flow can be studied directly in nature in ring species that comprise two reproductively isolated populations connected by a chain or ring of intergrading populations. We applied three tiers of spatio-temporal analysis (phylogeny/historical biogeography, phylogeography and landscape/population genetics) to the data from mitochondrial and nuclear genomes of eastern Australian parrots of the Crimson Rosella Platycercus elegans complex to understand the history and present genetic structure of the ring they have long been considered to form. A ring speciation hypothesis does not explain the patterns we have observed in our data (e.g. multiple genetic discontinuities, discordance in genotypic and phenotypic assignments where terminal differentiates meet). However, we cannot reject that a continuous circular distribution has been involved in the group's history or indeed that one was formed through secondary contact at the 'ring's' east and west; however, we reject a simple ring-species hypothesis as traditionally applied, with secondary contact only at its east. We discuss alternative models involving historical allopatry of populations. We suggest that population expansion shown by population genetics parameters in one of these isolates was accompanied by geographical range expansion, secondary contact and hybridization on the eastern and western sides of the ring. Pleistocene landscape and sea-level and habitat changes then established the birds' current distributions and range disjunctions. Populations now show idiosyncratic patterns of selection and drift. We suggest that selection and drift now drive evolution in different populations within what has been considered the ring.     

Mannose-binding lectin (MBL) facilitates opsonophagocytosis of yeasts but not of bacteria despite MBL binding

Mannose-binding lectin (MBL) is a serum protein of the innate immune system. After binding to a microorganism, MBL in complex with MBL-associated serine proteases activates the complement system, resulting in cleavage of complement factor C3. Cleaved C3 on the surface of the microorganism mediates opsonization for clearance, but the impact of MBL on subsequent phagocytosis has not been widely studied. We investigated the role of MBL in complement activation and phagocytosis of various bacteria and yeast species by flow cytometry. We measured both the C3 deposition during serum opsonization of fluorescent-labeled microorganisms as well as subsequent uptake of these microorganisms by human neutrophils. In MBL-deficient sera, a consistently decreased C3 deposition on both zymosan and Candida albicans was found and a reduced phagocytosis by neutrophils that was restored by exogenous MBL. This indicates that the lectin pathway of complement activation is important for the opsonophagocytosis of yeasts. In contrast, the C1q-dependent classical pathway dominated in the opsonization and phagocytosis of Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, whereas no effect of MBL was found. Both the lectin and the classical pathway of complement activation were highly amplified by the alternative route for opsonophagocytosis by neutrophils of yeast as well as microbial species. In summary, our data demonstrate that yeast species are preferentially opsonized and subsequently phagocytosed via activation of the lectin pathway of complement, whereas the uptake of bacterial strains was found to be largely MBL independent.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.     

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.     

Enhanced efficacy of anti-D immunoglobulin for treating ITP is not explained by higher immunoglobulin polymer content.

Several reports have suggested that low dose anti-D immunoglobulin is superior to high dose immunoglobulin for treatment of idiopathic thrombocytopenia purpura (ITP). However, some findings suggest that it is not the anti-D activity per se that is responsible for efficacy for treatment of ITP with anti-D immunoglobulin. Amongst alternative explanations for the mechanism of action is a relatively higher immunoglobulin polymer content of anti-D compared to other immunoglobulin products, which is more efficient in causing reticuloendothelial Fc receptor blockade. In order to investigate this we have evaluated the polymeric IgG content of anti-D and other immunoglobulin products. Different products showed considerable variation in immunoglobulin polymer content. There was no clear correlation between aggregate or dimer content and product type and anti-D as a class of product did not contain higher amounts of either dimer or aggregate compared to other products. Some manufacturers' products increased in polymer content on storage, but others did not show this effect. Therefore, higher immunoglobulin dimer and/or aggregate content cannot explain the increased efficacy of anti-D immunoglobulin for treatment of ITP. The role of polymeric Ig in efficacy for treatment of ITP is unclear.     

Productivity overshadows temperature in determining soil and ecosystem respiration across European forests

This paper presents CO₂ flux data from 18 forest ecosystems, studied in the European Union funded EUROFLUX project. Overall, mean annual gross primary productivity (GPP, the total amount of carbon (C) fixed during photosynthesis) of these forests was 1380 ± 330 gC m^?2 y^?1 (mean ±SD). On average, 80% of GPP was respired by autotrophs and heterotrophs and released back into the atmosphere (total ecosystem respiration, TER = 1100 ± 260 gC m^?2 y^?1). Mean annual soil respiration (SR) was 760 ± 340 gC m^?2 y^?1 (55% of GPP and 69% of TER). Among the investigated forests, large differences were observed in annual SR and TER that were not correlated with mean annual temperature. However, a significant correlation was observed between annual SR and TER and GPP among the relatively undisturbed forests. On the assumption that (i) root respiration is constrained by the allocation of photosynthates to the roots, which is coupled to productivity, and that (ii) the largest fraction of heterotrophic soil respiration originates from decomposition of young organic matter (leaves, fine roots), whose availability also depends on primary productivity, it is hypothesized that differences in SR among forests are likely to depend more on productivity than on temperature. At sites where soil disturbance has occurred (e.g. ploughing, drainage), soil espiration was a larger component of the ecosystem C budget and deviated from the relationship between annual SR (and TER) and GPP observed among the less‐disturbed forests. At one particular forest, carbon losses from the soil were so large, that in some years the site became a net source of carbon to the atmosphere. Excluding the disturbed sites from the present analysis reduced mean SR to 660 ± 290 gC m^?2 y^?1, representing 49% of GPP and 63% of TER in the relatively undisturbed forest ecosystems.