DNA Pyrosequencing Research Articles

Towards molecular biomarkers for biogas production from lignocellulose-rich substrates

Biogas production from lignocellulose-rich agricultural residues is gaining increasingly importance in sustainable energy production. Hydrolysis/acidogenesis (H/A) of lignocellulose as the initial rate-limiting step deserves particular optimization. A mixture of straw/hay was methanized applying two-phase digester systems with an initial H/A reactor and a one-stage system at different, meso- and thermophilic temperatures. H/A was intensified with increasing pH values and increasing temperature. H/A fermenters, however, were prone to switch to methanogenic systems at these conditions. Substrate turnover was accelerated in the bi-phasic process but did not reach the methanation efficiency of the single-stage digestion. There was no indication that two different cellulolytic inocula could establish in the given process.Bacterial communities were analyzed applying conventional amplicon clone sequencing targeting the hypervariable 16S rRNA gene region V6–V8 and by metagenome analyses applying direct DNA pyrosequencing without a PCR step. Corresponding results suggested that PCR did not introduce a bias but offered better phylogenetic resolution. Certain Clostridium IV and Prevotella members were most abundant in the H/A system operated at 38 °C, certain Clostridium III and Lachnospiraceae bacteria in the 45 °C, and certain Clostridium IV and Thermohydrogenium/Thermoanaerobacterium members in the 55 °C H/A system. Clostridium III representatives, Lachnospiraceae and Thermotogae dominated in the thermophilic single-stage system, in which also a higher portion of known syntrophic acetate oxidizers was found.Specific (RT-)qPCR systems were designed and applied for the most significant and abundant populations to assess their activity in the different digestion systems. The RT-qPCR results agreed with the DNA based community profiles obtained at the different temperatures. Up to 1012 16S rRNA copies mL−1 were determined in H/A fermenters with prevalence of rRNA of a Ruminococcaceae subgroup. Besides, Thermohydrogenium/Thermoanaerobacterium rRNA prevailed at thermophilic and Prevotellaceae rRNA at mesophilic conditions. The developed (RT)-qPCR systems can be used as biomarkers to optimize biogas production from straw/hay and possibly other lignocellulosic substrates.

Methylation and gene expression responses to ethanol feeding and betaine supplementation in the cystathionine beta synthase-deficient mouse.

Alcoholic steatohepatitis (ASH) is caused in part by the effects of ethanol (EtOH) on hepatic methionine metabolism. To investigate the phenotypic and epigenetic consequences of altered methionine metabolism in this disease, we studied the effects of 4-week intragastric EtOH feeding with and without the methyl donor betaine in cystathionine beta synthase (CβS) heterozygous C57BL/6J mice. The histopathology of early ASH was induced by EtOH feeding and prevented by betaine supplementation, while EtOH feeding reduced and betaine supplementation maintained the hepatic methylation ratio of the universal methyl donor S-adenosylmethionine (SAM) to the methyltransferase inhibitor S-adenosylhomocysteine (SAH). MethylC-seq genomic sequencing of heterozygous liver samples from each diet group found 2 to 4% reduced methylation in gene bodies, but not promoter regions of all autosomes of EtOH-fed mice, each of which were normalized in samples from mice fed the betaine-supplemented diet. The transcript levels of nitric oxide synthase (Nos2) and DNA methyltransferase 1 (Dnmt1) were increased, while those of peroxisome proliferator receptor-α (Pparα) were reduced in EtOH-fed mice, and each was normalized in mice fed the betaine-supplemented diet. DNA pyrosequencing of CβS heterozygous samples found reduced methylation in a gene body of Nos2 by EtOH feeding that was restored by betaine supplementation and was correlated inversely with its expression and positively with SAM/SAH ratios. The present study has demonstrated relationships among EtOH induction of ASH with aberrant methionine metabolism that was associated with gene body DNA hypomethylation in all autosomes and was prevented by betaine supplementation. The data imply that EtOH-induced changes in selected gene transcript levels and hypomethylation in gene bodies during the induction of ASH are a result of altered methionine metabolism that can be reversed through dietary supplementation of methyl donors.

Isolation and characterisation of 16 microsatellite markers for the endangered Gould’s long-eared bat (Nyctophilus gouldi) and cross-amplification in the lesser long-eared bat (N. geoffroyi)

Sixteen microsatellite markers were developed for use on two species of long-eared bats (Nyctophilus). 454 pyrosequencing of genomic DNA was conducted on N. gouldi which is listed as endangered in South Australia. Fifteen loci successfully amplified on N. gouldi while nine cross-amplified for use on N. geoffroyi. Two populations from south-eastern Australia were genotyped for each species, comprising 91 individuals for N. gouldi and 70 individuals for N. geoffroyi. There was no evidence of linkage disequilibrium and all loci displayed Hardy–Weinberg equilibrium except Nyg19 and Nyg39 which displayed evidence of null alleles in both N. geoffroyi populations. These markers will prove valuable in assessing connectivity between endangered populations of N. gouldi, and facilitate a comparative investigation into the impacts of habitat fragmentation on two vespertilionids.

Xylooligosaccharide increases bifidobacteria but not lactobacilli demonstrating potential for obesity prevention and treatment (37.6)

Background: Prebiotics can alter gut bacteria in obese subjects towards patterns seen in lean individuals.Objective: This study was conducted to determine the tolerance and effects of xylooligosaccharide (XOS) on human colonic microbiota, pH and short chain fatty acids (SCFA).Design: Healthy adult subjects (n=32) were randomized to receive 1.4 g, 2.8 g XOS or placebo in a 12 week study consisted of a 2‐week run‐in, an 8‐week intervention,and a 2‐week washout. Stool samples were collected at baseline, after 4 and 8 weeks of intervention and 2 weeks after the intervention for culture, pyrosequencing of community DNA, pH and SCFA analyses.Results: XOS was tolerated without significant gastrointestinal side effects. Both XOS groups achieved increases in Bifidobacterium counts compared to the placebo subjects, with the 2.8 g/day group showing significantly greater increases than the 1.4g/day group. Total anaerobic counts and Bacteroides fragilis group counts were significantly higher in the 2.8 g/day XOS group. There were no significant differences in the counts of Lactobacillus, Enterobacteriaceae and Clostridium between the three groups.XOS had no significant effect on stool pH, SCFA or lactic acid. Pyrosequencing showed no notable shifts in bacterial diversity.Conclusions: XOS supplementation may be beneficial to gastrointestinal microbiota and 2.8 g/day may be more effective than 1.4 g/day.

Epigenetic Regulation of Vitamin D Metabolism in Human Lung Adenocarcinoma

1α,25-Dihydroxyvitamin D3 (1,25-D3) is antiproliferative in preclinical models of lung cancer, but in tumor tissues, its efficacy may be limited by CYP24A1 expression. CYP24A1 is the rate limiting catabolic enzyme for 1,25-D3 and is overexpressed in human lung adenocarcinoma (AC) by unknown mechanisms. The DNA methylation status of CYP24A1 was determined by bisulfite DNA pyrosequencing in a panel of 30 lung cell lines and 90 surgically resected lung AC. The level of CYP24A1 methylation was correlated with CYP24A1 expression in lung AC cell lines and tumors. In addition, histone modifications were assessed by quantitative chromatin immunoprecipitation-polymerase chain reaction (ChIP-qPCR) in A549, NCI-H460, and SK-LU-1. Bisulfite DNA pyrosequencing analysis revealed that CYP24A1 gene was heterogeneously methylated in lung AC. Expression of CYP24A1 was inversely correlated with promoter DNA methylation in lung AC cell lines and tumors. Treatment with 5-aza-2'-deoxycytidine (5-Aza) and trichostatin A (TSA) increased CYP24A1 expression in lung AC. We observed that CYP24A1 promoter hypermethylation decreased CYP24A1 enzyme activity in vitro, whereas treatment with 5-Aza and/or TSA increased CYP24A1 enzyme affinity for its substrate 1,25-D3. In addition, ChIP-qPCR analysis revealed specific histone modifications within the CYP24A1 promoter region. Treatment with TSA increased H3K4me2 and H3K9ac and simultaneously decreased H3K9me2 at the CYP24A1 promoter and treatment with 5-Aza and/or TSA increased the recruitment of vitamin D receptor (VDR) to vitamin D response elements (VDRE) of the CYP24A1 promoter. The expression of CYP24A1 gene in human lung AC is in part epigenetically regulated by promoter DNA methylation and repressive histone modifications. These findings should be taken into consideration when targeting CYP24A1 to optimize antiproliferative effects of 1,25-D3 in lung AC.

Pyrosequencing of prey DNA in faeces of carnivorous land snails to facilitate ecological restoration and relocation programmes

Identifying and understanding predator diets is of high importance in biological conservation. This is particularly true for the introduction, establishment and maintenance of predator populations in newly created or modified ecological communities, such as translocation sites or restored habitats. Conservation status of predators may not permit captive feeding trials or intrusive gut-content methods, so non-intrusive diet assessment is required, such as faecal analysis. However, prey such as earthworms leave no morphological clues suitable for accurately discriminating between species consumed through visual faecal analysis. This study uses non-intrusive molecular methods on earthworm DNA extracted from the faeces of the carnivorous land snail Powelliphanta patrickensis to identify its earthworm diet and any seasonal trends. Data from 454-pyrosequencing revealed earthworm DNA in all samples (n = 60). Sequences were compared to a DNA library created from published and unpublished studies of New Zealand's endemic earthworms and online databases. Unidentified earthworm sequences were clustered into molecular operational taxonomic units (MOTUs). Twenty-six MOTUs were identified, 17 of which matched the library, whereas nine did not. Similarity indices indicate that there were seasonal differences (P < 0.05) in the earthworm communities represented in the summer and the winter diets. This study highlights the importance of utilising the vast body of data generated by pyrosequencing to investigate potential temporal diet shifts in protected species. The method described here is widely applicable to a wide range of predatory species of conservation interest and can further inform habitat restoration and relocation programmes to optimize the long-term survival of the target species.

Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

BackgroundSmall liver and minute intestinal flukes are highly prevalent in Southeast Asia. Definitive diagnosis of parasite infection is usually achieved parasitologically by finding the fluke eggs in feces. However, their eggs are difficult to differentiate morphologically in fecal samples, even for experienced technicians. The present study developed a PCR assay coupled with DNA pyrosequencing for identification of the fish-borne trematodes (FBT), Opisthorchis viverrini, Clonorchis sinensis, Haplorchis taichui, H. pumilio and Stellantchasmus falcatus, and to evaluate potential detection in fecal specimens, and identification and differentiation of cercarial and metacercarial stages.MethodsPrimers targeting the partial 28S large subunit ribosomal RNA gene were designed and about 46–47 nucleotides were selected as the target region for species identification by a PCR assay coupled with a pyrosequencing technique.ResultsThe nucleotide variations at 24 positions, which is sufficient for the identification of the five species of FBT were selected. The method could identify O. viverrini and C. sinensis eggs in feces, cercarial and metacercarial stages of O. viverrini, and metacercarial stage of H. pumilio and H. taichui. The detection limit was as little as a single O. viverrini or C. sinensis egg artificially inoculated in 100 mg of non-infected fecal sample (equivalent to 10 eggs per gram), indicating highly sensitivity. The method was found to be superior to the traditional microscopy method and was more rapid than Sanger DNA sequencing.ConclusionsDNA pyrosequencing-based identification is a valuable tool for differentiating O. viverrini and other Opisthorchis-like eggs, and can be applied to epidemiological studies and for molecular taxonomic investigation of FBT in endemic areas.

Effect of feeding dried distillers grains with solubles on ruminal biohydrogenation, intestinal fatty acid profile, and gut microbial diversity evaluated through DNA pyro-sequencing

The objectives of this study were to evaluate the effect of dried distillers grains with solubles (DDGS) on ruminal biohydrogenation and duodenal flow of fatty acids, and to evaluate effects on the ruminal and duodenal microbial community using Roche 454 pyro-sequencing. Three crossbred steers (average BW 780 ± 137 kg) fitted with ruminal and duodenal cannulae were used in a 3-diet, 6-period crossover design. Animals were housed in individual free stalls and fed twice daily at 0700 and 1900 h. Diets (DM basis) were 1) CONTROL, 19.5% corn bran, 20% sorghum silage, 60% brome hay, 0.5% trace minerals, and 0.25% urea, but no DDGS; 2) LOW DDGS, inclusion of 9.75% DDGS replacing equal percentage of corn bran; 3) HIGH DDGS, inclusion of 19.5% DDGS completely replacing corn bran. Feed ingredients and duodenal digesta samples were analyzed for fatty acid composition. The DNA was extracted from isolated mixed ruminal bacterial samples and from intestinal digesta samples. The V1-V3 region of the 16S rRNA gene was sequenced, and bacterial phylogenetic analysis was conducted. Data were analyzed using the MIXED procedure of SAS. Biohydrogenation of C18:1 increased (P < 0.01) with DDGS inclusion; means were 68.3, 75.6, and 79.3 ± 4.3% for CONTROL, LOW DDGS, and HIGH DDGS, respectively. In the same order, means of biohydrogenation of C18:2 (P < 0.05) were 84.1, 91.5, and 93.3 ± 3.4%. Duodenal flow of total fatty acids increased (P < 0.01) with DDGS inclusion; means were 134, 168, and 223 ± 33 g/d for CONTROL, LOW DDGS, and HIGH DDGS, respectively. In the same order, means of C18:0 flow (P < 0.01) were 51, 86, and 121 ± 18 g/d. DDGS did not affect the predominant bacterial phyla in the gut, which were Bacteroidetes (P = 0.62) and Firmicutes (P = 0.71). However, the phylum Fibrobacteres decreased (P < 0.01) when DDGS was fed with means of 5.5, 6.0 and 3.7 ± 0.6% for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Fibrobacteres were lower (P < 0.01) in isolated ruminal bacterial samples compared to duodenal digesta samples with means of 0.1 and 10.1 ± 0.6%, respectively. Overall, the inclusion of DDGS in diets increased ruminal biohydrogenation of C18:1 and C18:2, which increased duodenal flow of C18:0. In addition, the bacterial community of the rumen clustered separately from that of the duodenum suggesting different bacterial diversity between isolated ruminal bacteria and duodenal digesta.

Comparison of bacterial community in aqueous and oil phases of water-flooded petroleum reservoirs using pyrosequencing and clone library approaches

Bacterial communities in both aqueous and oil phases of water-flooded petroleum reservoirs were characterized by molecular analysis of bacterial 16S rRNA genes obtained from Shengli Oil Field using DNA pyrosequencing and gene clone library approaches. Metagenomic DNA was extracted from the aqueous and oil phases and subjected to polymerase chain reaction amplification with primers targeting the bacterial 16S rRNA genes. The analysis by these two methods showed that there was a large difference in bacterial diversity between the aqueous and oil phases of the reservoir fluids, especially in the reservoirs with lower water cut. At a high phylogenetic level, the predominant bacteria detected by these two approaches were identical. However, pyrosequencing allowed the detection of more rare bacterial species than the clone library method. Statistical analysis showed that the diversity of the bacterial community of the aqueous phase was lower than that of the oil phase. Phylogenetic analysis indicated that the vast majority of sequences detected in the water phase were from members of the genus Arcobacter within the Epsilonproteobacteria, which is capable of degrading the intermediates of hydrocarbon degradation such as acetate. The oil phase of reservoir fluid samples was dominated by members of the genus Pseudomonas within the Gammaproteobacteria and the genus Sphingomonas within the Alphaproteobacteria, which have the ability to degrade crude oil through adherence to hydrocarbons under aerobic conditions. In addition, many anaerobes that could degrade the component of crude oil were also found in the oil phase of reservoir fluids, mainly in the reservoir with lower water cut. These were represented by Desulfovibrio spp., Thermodesulfovibrio spp., Thermodesulforhabdus spp., Thermotoga spp., and Thermoanaerobacterium spp. This research suggested that simultaneous analysis of DNA extracted from both aqueous and oil phases can facilitate a better understanding of the bacterial communities in water-flooded petroleum reservoirs.

Xylooligosaccharide increases bifidobacteria but not lactobacilli in human gut microbiota

This study was conducted to determine the tolerance and effects of the prebiotic xylooligosaccharide (XOS) on the composition of human colonic microbiota, pH and short chain fatty acids (SCFA) in order to determine whether significant changes in the microbiota would be achievable without side effects. Healthy adult subjects (n = 32) were recruited in a double-blind, randomized, placebo-controlled study. Subjects received 1.4 g XOS, 2.8 g XOS or placebo in daily doses. The study consisted of a 2 week run-in, an 8 week intervention, and a 2 week washout phase. Stool samples were collected at baseline, after 4 and 8 weeks of intervention and 2 weeks after cessation of intervention. Samples were subjected to culture, pyrosequencing of community DNA, pH and SCFA analyses. Tolerance was evaluated by daily symptom charts. XOS was tolerated without significant gastrointestinal side effects. Bifidobacterium counts increased in both XOS groups compared to the placebo subjects, the 2.8 g per day group showed significantly greater increases than the 1.4 g per day group. Total anaerobic counts and Bacteroides fragilis group counts were significantly higher in the 2.8 g per day XOS group. There were no significant differences in the counts of Lactobacillus, Enterobacteriaceae and Clostridium between the three groups. XOS intervention had no significant effect on stool pH, SCFA or lactic acid. Pyrosequencing showed no notable shifts in bacterial diversity. XOS supplementation may be beneficial to gastrointestinal microbiota and 2.8 g per day may be more effective than 1.4 g per day. The low dose required and lack of gastrointestinal side effects makes the use of XOS as a food supplement feasible.

GCKGene-Body Hypomethylation Is Associated with the Risk of Coronary Heart Disease

Objectives. Glucokinase encoded by GCK is a key enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Variants of GCK gene were shown to be associated with type 2 diabetes (T2D) and coronary heart disease (CHD). The goal of this study was to investigate the contribution of GCK gene-body methylation to the risk of CHD. Design and Methods. 36 patients (18 males and 18 females) and 36 age- and sex-matched controls were collected for the current methylation research. DNA methylation level of the CpG island (CGI) region on the GCK gene-body was measured through the sodium bisulfite DNA conversion and pyrosequencing technology. Results. Our results indicated that CHD cases have a much lower methylation level (49.77 ± 6.43%) compared with controls (54.47 ± 7.65%, P = 0.018). In addition, GCK gene-body methylation was found to be positively associated with aging in controls (r = 0.443, P = 0.010). Conclusions. Our study indicated that the hypomethylation of GCK gene-body was significantly associated with the risk of CHD. Aging correlates with an elevation of GCK methylation in healthy controls.

DNA methylation of the MAPT gene in Parkinson's disease cohorts and modulation by vitamin E In Vitro

Parkinson's disease (PD) is a neurodegenerative disorder for which environmental factors influence disease risk and may act via an epigenetic mechanism. The microtubule-associated protein tau (MAPT) is a susceptibility gene for idiopathic PD. Methylation levels were determined by pyrosequencing of bisulfite-treated DNA in a leukocyte cohort (358 PD patients and 1084 controls) and in two brain cohorts (Brain1, comprising 69 cerebellum controls; and Brain2, comprising 3 brain regions from 28 PD patients and 12 controls). In vitro assays involved the transfection of methylated promoter-luciferase constructs or treatment with an exogenous micronutrient. In normal leukocytes, the MAPT H1/H2 diplotype and sex were predictors of MAPT methylation. Haplotype-specific pyrosequencing confirmed that the H1 haplotype had higher methylation than the H2 haplotype in normal leukocytes and brain tissues. MAPT methylation was negatively associated with MAPT expression in the Brain1 cohort and in transfected cells. Methylation levels differed between three normal brain regions (Brain2 cohort, putamen < cerebellum < anterior cingulate cortex). In PD samples, age at onset was positively associated with MAPT methylation in leukocytes. Moreover, there was hypermethylation in the cerebellum and hypomethylation in the putamen of PD patients compared with controls (Brain2 cohort). Finally, leukocyte methylation status was positively associated with blood vitamin E levels, and the effect was more significant in H2 haplotype carriers; this result was confirmed in cells that were exposed to 100 μM vitamin E. The significant effects of sex, diplotype, and brain region suggest that hypermethylation of the MAPT gene is neuroprotective by reducing MAPT expression. The effect of vitamin E on MAPT represents a possible gene-environment interaction.

Tag-encoded pyrosequencing analysis of bacterial diversity within different alpine grassland ecosystems of the Qinghai-Tibet Plateau, China

The Qinghai-Tibet Plateau is sensitive to climate change, with ecosystems that are important with respect to scientific research. Here high-throughput DNA pyrosequencing was used to assess bacterial diversity within different alpine grassland ecosystems of the Qinghai-Tibet Plateau, China. In total, 34,759 sequences were obtained for the three ecosystems––alpine cold swamp meadow (ASM), alpine cold meadow (AM), alpine sandy grassland (ASG), and 31 phyla and a small number of unclassified bacteria were detected. The bacterial community structures were different for each alpine grassland ecosystem. The Proteobacteria and Acidobacteria were the predominant phyla in all three ecosystems. Besides this, Actinobacteria and Chloroflexi were abundant in ASM, Bacteroidetes, Gemmatimonadetes and Verrucomicrobia were abundant in AM, and Actinobacteria were abundant in ASG. In addition, the functional bacterial genera also differed with each alpine grassland ecosystem. The ASM contained more nitrifying bacteria, methane-oxidizing bacteria and sulfur- and sulfate-reducing bacteria, whereas the ASG ecosystem contained more nitrogen-fixing bacteria. Pyrosequencing provided a greater insight into bacterial diversity within different alpine grassland ecosystems than previously possible, and gave key data for the involvement of bacteria in the protection of alpine grassland ecosystems of the Qinghai-Tibet Plateau, China.

Characterisation of the soil bacterial community structure and composition of natural and constructed wetlands

In the present study, the pyrosequencing of 16S ribosomal DNA was used to characterise the soil bacterial community composition of a constructed wetland receiving municipal wastewater and a nearby natural wetland. Soil samples were taken from different locations in each wetland (lagoon, zone with T. latifolia, zone with S. atrocinerea). Moreover, the water quality parameters were evaluated (pH, Tª, conductivity, dissolved oxygen, redox potential, nutrients and suspended solids), revealing that the organic matter and nutrient contents were significantly higher in the constructed wetland than in the natural one. In general, the bacterial communities of the natural wetland were more diverse than those of the constructed wetland. The major phylogenic groups of all soils included Proteobacteria, Verrucomicrobia and Chloroflexi, with Proteobacteria being the majority of the community composition. The Verrucomicrobia and Chloroflexi phyla were more abundant in the natural wetland than the constructed wetland; in contrast, the Proteobacteria phylum was more abundant in the constructed wetland than the natural wetland. Beta diversity analyses reveal that the soil bacterial communities in the natural wetland were less dissimilar to each other than to those of the constructed wetland.

Monitoring the rumen pectinolytic bacteriaTreponema saccharophilumusing real-time PCR

Treponema saccharophilum is a pectinolytic bacterium isolated from the bovine rumen. The abundance of this bacterium has not been well determined, reflecting the lack of a reliable and accurate detection method. To develop a rapid method for monitoring T.saccharophilum, we performed pyrosequencing of genomic DNA isolated from rumen microbiota to explore the 16S rRNA gene sequences of T.saccharophilum candidates. Species-specific primers were designed based on fifteen sequences of partial 16S rRNA genes generated through pyrosequencing with 97% or higher similarity with T.saccharophilum DSM2985 along with sequence from type strain. The relative abundance of T.saccharophilum was quantified in both in vitro and in vivo rumen systems with varied pectin-containing forages using real-time PCR. There was a clear association of T.saccharophilum with alfalfa hay, which contains more pectin than Chinese wild rye hay or corn stover. The relative abundance of T.saccharophilum was as high as 0.58% in vivo, comparable with the population density of other common rumen bacteria. It is recognized that T.saccharophilum plays an important role in pectin digestion in the rumen.

Strong functional stability of soil microbial communities under semiarid Mediterranean conditions and subjected to long-term shifts in baseline precipitation

We investigated the effect of soil microclimate on the structure and functioning of soil microbial communities in a Mediterranean Holm-oak forest subjected to 10 years of partial rain exclusion manipulations, simulating average drought conditions expected in Mediterranean areas for the following decades. We applied a high throughput DNA pyrosequencing technique coupled to parallel measurements of microbial respiration (RH) and temperature sensitivity of microbial respiration (Q10). Some consistent changes in the structure of bacterial communities suggest a slow process of community shifts parallel to the trend towards oligotrophy in response to long-term droughts. However, the structure of bacterial communities was mainly determined by short-term environmental fluctuations associated with sampling date (winter, spring and summer) rather than long-term (10 years) shifts in baseline precipitation. Moreover, long-term drought did not exert any chronic effect on the functioning of soil microbial communities (RH and Q10), emphasizing the functional stability of these communities to this long-term but mild shifts in water availability. We hypothesize that the particular conditions of the Mediterranean climate with strong seasonal shifts in both temperature and soil water availability but also characterized by very extreme environmental conditions during summer, was acting as a strong force in community assembling, selecting phenotypes adapted to the semiarid conditions characterizing Mediterranean ecosystems. Relations of climate with the phylogenetic structure and overall diversity of the communities as well as the distribution of the individual responses of different lineages (genera) to climate confirmed our hypotheses, evidencing communities dominated by thermotolerant and drought-tolerant phenotypes.

Patients Treated With Decitabine Demonstrate Changes In β-Catenin Localization From The Nucleus To The Cytoplasm In Circulating Blasts

BackgroundThe DNA methyltransferase inhibitors (DNMTi) 5-azacitidine (Aza) and decitabine (Dac) are a standard of care for patients with myelodysplatic syndrome (MDS) and acute myeloid leukemia (AML). Many hypotheses exist for the mechanism of these agents, including re-expression of epigenetically silenced tumor suppressor genes and direct cytotoxicity. We and others have shown hypermethylation of WNT/β-catenin inhibitory genes in primary MDS and AML samples. Activation of WNT/β-catenin signaling has furthermore been shown to play a role in the development and relapse of AML. Inhibition of WNT/β-catenin signaling is proposed to be clinically beneficial in AML. Based on published and preliminary data demonstrating that AML cell lines exposed to Aza and Dac re-express WNT/b-catenin inhibitors and down-regulate WNT/β-catenin signaling, we hypothesized that patients with AML receiving Dac would demonstrate suppression of WNT/β-catenin signaling in circulating blasts. MethodsWe obtained serial peripheral blood samples from 5 patients with AML during their first cycle of Dac therapy (20 mg/m2 per day for 10 days). These patients were ineligible for high-dose induction chemotherapy based on age, performance status or comorbid illness and were of intermediate or poor risk karyotype. All patients had circulating blasts at presentation, which remained stable or declined over time. Blast populations were identified using flow cytometric selection based upon the characteristics of the diagnostic sample. Global methylation was assessed (using LINE-1 as a surrogate) by bisulfite pyrosequencing of mononuclear cell DNA extracted from the peripheral blood. To determine whether reduced methylation was associated with decreases in WNT/β-catenin signaling, we performed imaging flow cytometry (ImageStream, Amnis Corporation) on viable blasts harvested pre-treatment and on days 5-9 of the first Dac cycle (at the methylation nadir). ImageStream was used to assess nuclear localization of β-catenin, a biochemical hallmark of active WNT signaling, in CD34+ blasts. Nuclear β-catenin was quantified using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). A resolution metric (Fishers discriminant ratio, Rd) was calculated to determine shifts in the population distributions of this similarity score between CD34+ blasts at diagnosis versus after Dac therapy; a negative Rd value indicates decreased similarity scores between β-catenin and DAPI in treated versus diagnostic samples and thus decreased nuclear β-catenin. To determine whether changes in nuclear β-catenin were associated with changes in gene transcription, we performed whole genome analysis of RNA extracted from diagnostic and follow up samples (n = 5 paired samples, Illumina Human-HT12v4 Expression BeadChip). ResultsAs expected, Dac treatment significantly reduced global DNA methylation (LINE-1 methylation) from 77.0 ± 1.6% pre-treatment to a nadir of 60.9 ± 6.8% (p< 0.01, n = 6). During the methylation nadir (days 5 to 9), the cellular distribution of β-catenin in CD34+ blasts shifted from the nucleus to the cytoplasm as indicated by an average Rd value of -0.22 (n = 5). This Rd value is similar to that observed after treating U937 cells with 0.5 µM Dac. The total amount of β-catenin in CD34+ blasts from treated patients did not change compared to their pre-treatment levels (0.98 ± 0.40 fold, n = 5) indicating that the shift in the cellular distribution of β-catenin from the nucleus towards the cytoplasm was not accompanied by decreased protein level. Comparison of samples from day 5-9 of Dac treatment to pre-treatment samples revealed 128 genes with a ≥ 1.2-fold change (75 up, 53 down, p< 0.05) in mRNA levels. We did not observe changes in expression of WNT regulatory genes, suggesting that alternative mechanisms may explain the reduction in nuclear β-catenin following Dac therapy. Of the 10 genes up-regulated more than 1.5-fold, 4 (FOS, EGR1, CLEC12A, RNASE2) are associated with myeloid differentiation. Of these, FOS and EGR1 are negatively regulated by WNT/β-catenin signaling. ConclusionsGiven that activation of WNT/β-catenin signaling can suppress hematopoietic differentiation, these data suggest a model in which Dac exposure reduces nuclear β-catenin, leading to increased expression of pro-differentiation genes. Disclosures:Wetzler:Teva: Honoraria, Membership on an entity's Board of Directors or advisory committees. Griffiths:Astex Pharmaceuticals: Research Funding; Celgene, Inc.: Honoraria; Alexion Pharmaceuticals: Honoraria.

Vertical Profiles of Bacteria in the Tropical and Subarctic Oceans Revealed by Pyrosequencing

Community composition of Bacteria in the surface and deep water layers were examined at three oceanic sites in the Pacific Ocean separated by great distance, i.e., the South China Sea (SCS) in the western tropical Pacific, the Costa Rica Dome (CRD) in the eastern tropical Pacific and the western subarctic North Pacific (SNP), using high throughput DNA pyrosequencing of the 16S rRNA gene. Bioinformatic analysis rendered a total of 143600 high quality sequences with an average 11967 sequences per sample and mean read length of 449 bp. Phylogenetic analysis showed that Proteobacteria dominated in all shallow and deep waters, with Alphaproteobacteria and Gammaproteobacteria the two most abundant components, and SAR11 the most abundant group at family level in all regions. Cyanobacteria occurred mainly in the surface euphotic layer, and the majority of them in the tropical waters belonged to the GpIIa family including Prochlorococcus and Synechococcus, whilst those associated with Cryptophytes and diatoms were common in the subarctic waters. In general, species richness (Chao1) and diversity (Shannon index H′) were higher for the bacterial communities in the intermediate water layers than for those in surface and deep waters. Both NMDS plot and UPGMA clustering demonstrated that bacterial community composition in the deep waters (500 m ∼2000 m) of the three oceanic regions shared a high similarity and were distinct from those in the upper waters (5 m ∼100 m). Our study indicates that bacterial community composition in the DOC-poor deep water in both tropical and subarctic regions were rather stable, contrasting to those in the surface water layers, which could be strongly affected by the fluctuations of environmental factors.

Changes of saliva microbiota in nasopharyngeal carcinoma patients under chemoradiation therapy

ObjectiveA growing body of evidence has implicated human oral microbiota in the aetiology of oral and systemic diseases. Nasopharyngeal carcinoma (NPC), an epithelial-originated malignancy, has a complex aetiology not yet fully understood. Chemoradiation therapy of NPC can affect oral microbiota and is usually accompanied by plaque accumulation. Thus, the study aimed to understand the diversity, divergence and development of the oral microbiota in NPC patients and their associated treatment, which might provide useful insights into disease aetiology and treatment side effects. DesignA longitudinal study was designed that included three Chinese adults with NPC. Saliva samples were collected at three time points: prior to the chemoradiation treatment (carcinoma baseline, or CB), 7 months post-treatment (carcinoma-after-therapy phase 1 or CA1) and 12 months post-treatment (carcinoma-after-therapy phase 2 or CA2). Pyrosequencing of the bacterial 16S ribosomal DNA (rDNA) V1–V3 hypervariable region was employed to characterise the microbiota. Saliva samples of three healthy subjects from our former study were employed as healthy controls. Principal coordinates analysis (PCoA), Metastats and random forest prediction models were used to reveal the key microbial members associated with NPC and its treatment programme. Results(1) In total, 412 bacterial species from at least 107 genera and 13 phyla were found in the saliva samples of the NPC patients. (2) PCoA revealed that not only were the microbiota from NPC patients distinct from those of healthy controls (p<0.001) but also that separation was found on the saliva microbiota between pre- and post-therapy (p<0.001) in the NPC samples. (3) At the genus level and the operational taxonomic unit (OTU) level, Streptococcus was found with lower abundance in NPC samples. (4) Chemoradiation therapy did not incur similar changes in microbiota structure among the three NPC patients; the microbiota in one of them stayed largely steady, while those in the other two showed significant alteration. ConclusionsThis is the first study employing culture-independent techniques to interrogate the phylogenetic diversity, divergence and temporal development of oral microbiota in NPC patients. Our results indicated that certain bacterial taxa might be associated with NPC and that oral microbiota of NPC patients might respond to the chemoradiation therapy in a host-specific manner. Further investigation with larger sample size should help to validate the links between oral microbiota and NPC.

A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects

The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25-92 years (mean = 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the ∼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 × 10(-8)). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10(-30)). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays.