Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrialanthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG, napA, and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (narG and napA) and nitrite reductase (nirS and nrfA) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

Quantitative polymerase chain reaction (Q-PCR) amplification is widely applied for determining gene and transcript numbers within environmental samples. This research evaluated Q-PCR reproducibility via TaqMan assays quantifying 16S rRNA gene and transcript numbers in sediments, within and between replicate Q-PCR assays. Intra-assay variation in 16S rRNA gene numbers in replicate DNA samples was low (coefficients of variation; CV from 3.2 to 5.2%). However, variability increased using replicated standard curves within separate Q-PCR assays (CV from 11.2% to 26%), indicating absolute comparison of gene numbers between Q-PCR assays was less reliable. 16S rRNA transcript quantification was evaluated using standard curves of diluted RNA or cDNA (before, or following, reverse transcription). These standard curves were statistically different with cDNA-derived curves giving higher r(2) values and Q-PCR efficiencies. Template concentrations used in Q-PCR also affected 16S rRNA gene and transcript numbers. For DNA, 10(-3) dilutions yielded higher gene numbers than 10(-1) and 10(-2) dilutions. Conversely, RNA template dilution reduced numbers of transcripts detected. Finally, different nucleic acid isolation methods also resulted in gene and transcript number variability. This research demonstrates Q-PCR determination of absolute numbers of genes and transcripts using environmental nucleic acids should be treated cautiously.

Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (narG, napA, nirS, and nrfA) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for ϳ30% of N 2 formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(n ؉ 1) process rate data and log(n ؉ 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.Estuaries are major conduits for the transport of anthropogenically derived nitrogen (e.g., from fertilizer runoff and from wastewater treatment plants) from land to sea (18,20). Estuarine sediments are now recognized as being an important location for the removal of inorganic nitrogen from this environment via benthic nitrate reduction to nitrogenous gases (21). Previously, we have utilized the isotope-pairing technique (22) to investigate rates of denitrification (DN) of nitrate to N 2 O and N 2 along the nitrate and salinity gradients in the hypernutrified Colne estuary, United Kingdom, and demonstrated that DN rates are highest at the estuary head, where nitrate levels are also at their highest (7, 9). However, DN represents only one of three key pathways relevant to nitrate reduction, with a recent review suggesting that the importance of DN in aquatic systems may be overstated (3). A substantial proportion of nitrate may alternatively be converted to ammonium (1, 16, 17) via dissimilatory nitrate reduction to ammonium (DNRA), in which case inorganic nitrogen is retained within the aquatic environment. Nitrite, usually derived from nitrate reduc...

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Background: The triglyceride glucose (TyG) index, a simple surrogate estimate of insulin resistance, has been demonstrated to predict cardiovascular (CV) disease morbidity and mortality in the general population and many patient cohorts. However, to our knowledge, the prognostic usefulness of the TyG index after percutaneous coronary intervention (PCI) in patients with type 2 diabetes mellitus (T2DM) and acute coronary syndrome (ACS) has not been determined. This study aimed to evaluate the association of the TyG index with adverse CV outcomes in patients with T2DM and ACS who underwent PCI. Methods: The TyG index was calculated using the formula ln[fasting triglycerides (mg/dL) × fasting glucose (mg/ dL)/2]. The primary endpoint was the composite of all-cause mortality, non-fatal stroke, non-fatal myocardial infarction, or unplanned repeat revascularization. The association between the TyG index and adverse CV outcomes was assessed by Cox proportional hazards regression analysis. Results: In total, 776 patients with T2DM and ACS who underwent PCI (mean age, 61 ± 10 years; men, 72.2%) were included in the final analysis. Over a median follow-up of 30 months, 188 patients (24.2%) had at least 1 primary endpoint event. The follow-up incidence of the primary endpoint rose with increasing TyG index tertiles. The multivariate Cox proportional hazards regression analysis adjusted for multiple confounders revealed a hazard ratio for the primary endpoint of 2.17 (95% CI 1.45-3.24; P for trend = 0.001) when the highest and lowest TyG index tertiles were compared. Conclusions: The TyG index was significantly and positively associated with adverse CV outcomes, suggesting that the TyG index may be a valuable predictor of adverse CV outcomes after PCI in patients with T2DM and ACS.

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We measured benthic denitrification (DN) and dissimilatory reduction of nitrate to ammonium (DNRA) using the isotope-pairing technique in three tropical estuaries in Thailand (Mae Klong), Indonesia (Cisadane), and Fiji (Vunidawa-Rewa) during rainy, dry, and intermediate seasons along the salinity gradient of each estuary. DNRA dominated. Anammox (AN) was measured initially but neither AN activity nor AN bacteria-related 16S ribosomal RNA genes were detected in any of the estuaries. DN was either zero or extremely low, driven by water column nitrate and not from benthic nitrification-DN. N 2 O was not formed during DN. N 2 O saturations in estuary water were low, except in the nutrified Indonesian estuary, and tropical estuaries are therefore likely to be only small sources of N 2 O. Benthic nitrate reduction was nitrate limited; when nitrate was enhanced experimentally, DN increased slightly, but DNRA increased proportionately much more. Predominance of DNRA over DN in tropical estuaries may be due both to an energetic advantage (greater standard free energy change, DGu) of nitrate ammonifiers over denitrifiers when competing for limited nitrate, and also to higher affinity for nitrate by the nitrate ammonifiers. At tropical temperatures the three processes occur in the order DNRA . DN . AN. In contrast, temperate estuaries, at lower temperature and higher nitrate concentrations, exhibit proportionately greater levels of AN and DN. The Cisadane estuary became anoxic during the dry season, with high ammonium and sulfide, but no nitrate reduction because of lack of nitrate. Addition of nitrate stimulated high rates of autotrophic DN driven by sulfide, but not DNRA.

Notoginseng (Panax notoginseng), a valuable herbal medicine, has high death rates in continuous cropping systems. Variation in the soil microbial community is considered the primary cause of notoginseng mortality, although the taxa responsible for crop failure remains unidentified. This study used high-throughput sequencing methods to characterize changes in the microbial community and screen microbial taxa related to the death rate. Fungal diversity significantly decreased in soils cropped with notoginseng for three years. The death rate and the fungal diversity were significantly negatively correlated, suggesting that fungal diversity might be a potential bioindicator of soil health. Positive correlation coefficients revealed that Burkholderiales, Syntrophobacteraceae, Myrmecridium, Phaeosphaeria, Fusarium, and Phoma were better adapted to colonization of diseased plants. The relative abundance of Fusarium oxysporum (R = 0.841, P < 0.05) and Phaeosphaeria rousseliana (R = 0.830, P < 0.05) were positively associated with the death rate. F. oxysporum was a pathogen of notoginseng root-rot that caused seedling death. Negative correlation coefficients indicated that Thermogemmatisporaceae, Actinosynnemataceae, Hydnodontaceae, Herpotrichiellaceae, and Coniosporium might be antagonists of pathogens, and the relative abundance of Coniosporium perforans was negatively correlated with the death rate. Our findings provide a dynamic overview of the microbial community and present a clear scope for screening beneficial microbes and pathogens of notoginseng.