Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO2

Elevated atmospheric CO₂ can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritus-based ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO₂ would result in changes in the size and composition of microbial communities colonizing leaves in a woodland stream. Three tree species, Populus tremuloides, Salix alba, and Acer saccharum, were grown under ambient (360 ppm) or elevated (720 ppm) CO₂, and their leaves were incubated in a woodland stream. Elevated-CO₂ treatment resulted in significant increases in the phenolic and tannin contents and C/N ratios of leaves. Microbial effects, which occurred only for P. tremuloides leaves, included decreased fungal biomass and decreased bacterial counts. Analysis of fungal and bacterial communities on P. tremuloides leaves via terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing revealed that fungal community composition was mostly unchanged by the elevated-CO₂ treatment, whereas bacterial communities showed a significant shift in composition and a significant increase in diversity. Specific changes in bacterial communities included increased numbers of alphaproteobacterial and cytophaga-flavobacter-bacteroides (CFB) group sequences and decreased numbers of betaproteobacterial and firmicutes sequences, as well as a pronounced decrease in overall Gram-positive bacterial sequences.The concentration of atmospheric CO₂ has been increasing for the last 150 years, from 270 ppm prior to the industrial revolution (49) to the current level of approximately 388 ppm (http://www.mlo.noaa.gov), and is projected to exceed 700 ppm by the end of the century (57). This ongoing increase in atmospheric CO₂ is believed to be due to the extensive use of fossil fuels and changes in land use patterns (5). Elevated atmospheric CO₂ has global climate implications due to its role in the greenhouse effect (39), and it has also been shown to have direct biological effects. Specifically, elevated CO₂ can increase the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco) (13), resulting in increased carbon fixation by C3 plants (49). This increased carbon fixation can result in increased above- and below-ground plant biomass (21, 47, 63, 72), as well as altered foliar chemical composition (31, 46, 58, 70).Elevated atmospheric CO₂ is unlikely to have direct impacts on forested headwater streams, as they are primarily heterotrophic systems (2) in which CO₂ is typically supersaturated (41). However, changes in leaf chemistry may have an impact, as forested headwater streams are detritus-based ecosystems that derive up to 99% of their carbon inputs from terrestrial organic matter (71), which is mainly leaf litter (29). Microbes play a key role in the entry of this allochthonous organic material into stream food webs. Fungi and bacteria colonize leaf litter after its deposition in a stream and begin decomposition of the leaf material (34). The resulting growth of microbial assemblages associated with leaf litter provides a critical food resource for detritus-feeding invertebrate consumers (6, 18, 23, 44), which through their feeding activities help facilitate the further transformation and breakdown of plant litter and the flow of carbon and nutrients to higher-trophic-level organisms, including fish. Prior research has demonstrated that aquatic invertebrates show a clear preference to eat leaves that have been extensively colonized, or “conditioned,” by microbes (4, 18, 65). This is likely due to the fact that microbial colonization significantly increases the nutrient content of detritus, as microbes can incorporate soluble nutrients from stream water (e.g., nitrogen) into the microbial biomass (64, 66). In addition, microbes convert indigestible leaf components (e.g., lignin and cellulose) into microbial biomass, which invertebrates can digest more efficiently (6). Therefore, fungi and bacteria are significant contributors to the transfer of carbon and nutrients from terrestrial to aquatic ecosystems.Microbial decomposition of leaves in streams is influenced by the chemical composition of the leaf material. This has been illustrated by a number of studies comparing decomposition of leaves from different tree species (for a review, see reference 62). These studies have demonstrated that leaves from species, such as oaks and conifers, that are relatively high in polyphenolic compounds, including lignin and tannins, tend to decompose more slowly than leaves from species with lower concentrations of these compounds, such as alder (62). The leaf carbon-to-nitrogen (C/N) ratio also impacts decomposition rates; leaf litter with a high C/N ratio tends to decompose more slowly than litter with a low C/N ratio (62). These trends are relevant to atmospheric CO₂ concentrations because elevated atmospheric CO₂ has been shown to increase the concentrations of phenolic compounds (lignin and tannins), as well as the C/N ratio of leaves of C3 plants (31, 46, 58, 70). Therefore, it is reasonable to hypothesize that growth of trees under elevated CO₂ could have negative impacts on microbial colonization and decomposition of leaves. Rier et al. (58) tested this hypothesis with one tree species, Populus tremuloides (quaking aspen), and found that leaves produced under elevated CO₂ decomposed more slowly in streams and supported less fungal and bacterial biomass than leaves produced under ambient conditions (58).In addition to impacting microbial community size, it is reasonable to hypothesize that changes in leaf chemistry caused by growth of trees under elevated CO₂ could impact microbial community composition. Several studies have demonstrated that the compositions of microbial communities colonizing leaves in streams can differ based on tree species (36, 45). No study we are aware of has examined the effects of tree growth under elevated atmospheric CO₂ on the compositions of microbial communities colonizing leaf litter in streams; however, such changes in microbial community composition could be highly relevant to stream food webs. For example, different groups of fungi and bacteria differ in their abilities to degrade various components of leaf litter (1, 67), so the species compositions of microbial communities could potentially impact rates of decomposition and production of microbial biomass (26). This in turn could impact the transfer of carbon and energy to higher-trophic-level organisms. In addition, different groups of fungi and bacteria differ in chemical composition (9, 32), and thus, they may differ in their nutritional values to aquatic invertebrates.In the current study, we tested the hypothesis that changes in leaf chemistry caused by elevated CO₂ would result in changes in the biomass and composition of detrital microbial communities by growing three tree species under ambient or elevated CO₂, collecting leaves after abscission, incubating the leaves in a woodland stream, and determining the biomass and composition of the microbial communities colonizing the leaves.     

An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.     

Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability

Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1-3 microg/mL. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a 12 microg/mL concentration (XTT assay). In the concentration range of 6-12 microg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.     

Patient-specific stem cell lines derived from human parthenogenetic blastocysts

Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.     

Impact of Obesity and Body Fat Distribution on Cardiovascular Risk Factors in Hong Kong Chinese

Objective: Body fat distribution has been reported to differentially contribute to the development of cardiovascular risk. We report the relative associations between general and central obesity and risk factors in 2893 Chinese subjects recruited from the Hong Kong population. Research Methods and Procedures: Anthropometric parameters [waist circumference (WC) and BMI], surrogate measures of insulin resistance (fasting plasma glucose and insulin, oral glucose tolerance test, 2 hours glucose and insulin), fasting lipids (total, low‐density lipoprotein‐cholesterol, high‐density lipoprotein‐cholesterol, and triglycerides) and systolic and diastolic blood pressure were measured. General obesity was classified as BMI ≥25.0 kg/m² and central obesity as a WC ≥80 or ≥90 cm in women and men, respectively. Results: A total of 39.2% of the population was found to be obese. Obesity per se increased the levels of the risk factors, but central adiposity contributed to a greater extent to adverse high‐density lipoprotein‐cholesterol, triglyceride, and insulin resistance levels. There was a continuous relationship between increasing obesity, both general and central, and cardiovascular risk, with lowest risk associated with the lowest indices of obesity. In the 1759 nonobese subjects divided into quartiles of BMI or WC, the levels of the cardiovascular risk factors still significantly increased with increasing quartiles of adiposity. Discussion: Central adiposity appears to contribute to a greater extent than general adiposity to the development of cardiovascular risk in this population. The relationship between obesity parameters and risk is a continuum, with risk factors significantly increasing even at levels usually considered nonobese. These observations support the proposed redefinition of overweight and obesity in Asian populations using lower cut‐off points.     

Plasma lipoprotein lipid and Lp[a] changes with substitution of elaidic acid for oleic acid in the diet.

The effect of additional dietary trans fatty acids (7% energy) on plasma lipids was assessed in a double-blind comparison of four separate diets: 1, enriched with butter fat (lauric-myristic-palmitic); 2, oleic acid-rich; 3, elaidic acid-rich; 4, palmitic acid-rich. The total dietary period was 11 weeks and comprised normal foods plus specific fat supplements. In 27 mildly hypercholesterolemic men, total and LDL cholesterol were significantly lower during the 3-week oleic acid-rich diet, and were similar during the other three diets. For the four diets LDL cholesterol levels were in mg/dl: 1, 163; 2, 151; 3, 165; 4, 161. HDL cholesterol was significantly higher with the palmitic acid-rich diet, 42 mg/dl, compared with elaidic acid, 38 mg/dl, which in turn was not lower than with oleic acid, 38 mg/dl. Plasma elaidic acid concentration rose seven-fold with the trans fatty acid diet but did not increase the vulnerability of LDL to oxidative change. The elaidic acid-rich diet led to significant elevations in the level of Lp[a] compared to all the other test diets. The Lp[a] level increased to 296 +/- 220 U/l in the elaidic acid-rich period from 235 +/- 182 (mean +/- SD) in the first ("butter") period (P less than 0.001) compared with 249 +/- 204 in the palmitic acid period (P less than 0.001) and 236 +/- 201 in the oleic acid period (NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Scaling-up from an implementation trial to state-wide coverage: results from the preliminary Melbourne Diabetes Prevention Study

ABSTRACT: BACKGROUND: The successful Greater Green Triangle Diabetes Prevention Program (GGT DPP), a small implementation trial, has been scaled-up to the Victorian state-wide 'Life!' programme with over 10,000 individuals enrolled. The Melbourne Diabetes Prevention Study (MDPS) is an evaluation of the translation from the GGT DPP to the Life! programme. We report results from the preliminary phase (pMDPS) of this evaluation. METHODS: The pMDPS is a randomised controlled trial with 92 individuals aged 50 to 75 at high risk of developing type 2 diabetes randomised to Life! or usual care. Intervention consisted of six structured 90-minute group sessions: five fortnightly sessions and the final session at 8 months. Participants underwent anthropometric and laboratory tests at baseline and 12 months, and provided self-reported psychosocial, dietary, and physical activity measures. Intervention group participants additionally underwent these tests at 3 months. Paired t tests were used to analyse within-group changes over time. Chi-square tests were used to analyse differences between groups in goals met at 12 months. Differences between groups for changes over time were tested with generalised estimating equations and analysis of covariance. RESULTS: Intervention participants significantly improved at 12 months in mean body mass index ([MINUS SIGN]0.98 kg/m2, standard error (SE) = 0.26), weight ([MINUS SIGN]2.65 kg, SE = 0.72), waist circumference ([MINUS SIGN]7.45 cm, SE = 1.15), and systolic blood pressure ([MINUS SIGN]3.18 mmHg, SE = 1.26), increased high-density lipoprotein-cholesterol (0.07 mmol/l, SE = 0.03), reduced energy from total ([MINUS SIGN]2.00%, SE = 0.78) and saturated fat ([MINUS SIGN]1.54%, SE = 0.41), and increased fibre intake (1.98 g/1,000 kcal energy, SE = 0.47). In controls, oral glucose at 2 hours deteriorated (0.59 mmol/l, SE = 0.27). Only waist circumference reduced significantly ([MINUS SIGN]4.02 cm, SE = 0.95).Intervention participants significantly outperformed controls over 12 months for body mass index and fibre intake. After baseline adjustment, they also showed greater weight loss and reduced saturated fat versus total energy intake.At least 5% weight loss was achieved by 32% of intervention participants versus 0% controls. CONCLUSIONS: pMDPS results indicate that scaling-up from implementation trial to state-wide programme is possible. The system design for Life! was fit for purpose of scaling-up from efficacy to effectiveness.Trial registrationAustralian and New Zealand Clinical Trials Registry ACTRN12609000507280.     

Sucrose Breakdown in Relation to Fruit Growth of Acid Lime (Citrus aurantifolia)

The physiological capacity for sucrose breakdown in developingjuice sac cells of acid limes was estimated by assaying theactivity of the three enzymes of sucrose catabolism in additionto vacuolar acid hydrolysis. The maximum potential rates ofsucrose breakdown were compared with the observed rates of carbonutilization. Highest potential rates of sucrose breakdown (28.621mmol cm^–3 per hydrated active space d^–1) occurredat the initial stages of fruit development where carbon utilizationwas highest. As the fruit developed, the potential rates ofsucrose breakdown and carbon utilization declined to very lowlevels. At 80% of development, vacuolar acid hydrolysis becamethe only physiological mechanism for sucrose breakdown. Therelatively low amounts of sucrose hydrolysed by acid hydrolysisat this time were just sufficient to account for the measuredcarbon demands. The results suggest that carbon supplied bythis distinct sucrose catabolizing system is able to provideadequate levels of carbon skeletons for the observed levelsof respiration and dry weight deposition early in development,but becomes a limiting factor for growth in the later stages. Key words: Vacuolar acid hydrolysis, Citrus aurantifolia     

Homing in on intracellular Abeta?

In this issue of Neuron, a study by Billings et al. points to intracellular Abeta as a possible cause of neuronal dysfunction. In a mouse model of Alzheimer's disease, Billings et al. link appearance of intraneuronal Abeta to cognitive impairments and then show that "clearance" of intraneuronal Abeta by anti-Abeta antibodies restores cognitive deficits.     

Culture conditions of Roseobacter strain 27-4 affect its attachment and biofilm formation as quantified by real-time PCR

The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions.