Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

Although parasuicidal behavior in adolescence is poorly understood, evidence suggests that it may be a developmental precursor of borderline personality disorder~BPD!. Current theories of both parasuicide and BPD suggest that emotion dysregulation is the primary precipitant of self-injury, which serves to dampen overwhelmingly negative affect. To date, however, no studies have assessed endophenotypic markers of emotional responding among parasuicidal adolescents. In the present study, we compare parasuicidal adolescent girls~n ϭ 23! with age-matched controls~n ϭ 23! on both psychological and physiological measures of emotion regulation and psychopathology. Adolescents, parents, and teachers completed questionnaires assessing internalizing and externalizing psychopathology, substance use, trait affectivity, and histories of parasuicide. Psychophysiological measures including electrodermal responding~EDR!, respiratory sinus arrhythmia, and cardiac pre-ejection period~PEP! were collected at baseline, during negative mood induction, and during recovery. Compared with controls, parasuicidal adolescents exhibited reduced respiratory sinus arrhythmia~RSA! at baseline, greater RSA reactivity during negative mood induction, and attenuated peripheral serotonin levels. No between-group differences on measures of PEP or EDR were found. These results lend further support to theories of emotion dysregulation and impulsivity in parasuicidal teenage girls.

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...

Terminal restriction fragment length polymorphism (tRFLP) is a potentially high-throughput method for the analysis of complex microbial communities. Comparison of multiple tRFLP profiles to identify shared and unique components of microbial communities however, is done manually, which is both time consuming and error prone. This paper describes a freely accessible web-based program, T-Align (http://inismor.ucd.ie/~talign/), which addresses this problem. Initially replicate profiles are compared and used to generate a single consensus profile containing only terminal restriction fragments that occur in all replicate profiles. Subsequently consensus profiles representing different communities are compared to produce a list showing whether a terminal restriction fragment (TRF) is present in a particular sample and its relative fluorescence intensity. The use of T-Align thus allows rapid comparison of numerous tRFLP profiles. T-Align is demonstrated by alignment of tRFLP profiles generated from bacterioplankton communities collected from the Irish and Celtic Seas in November 2000. Ubiquitous TRFs and site-specific TRFs were identified using T-Align.

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.

Self-inflicted injury in adolescence indicates significant emotional and psychological suffering. Although data on the etiology of self-injury are limited, current theories suggest that the emotional lability observed among self-injuring adolescents results from complex interactions between individual biological vulnerabilities and environmental risk. For example, deficiencies in serotonergic functioning, in conjunction with certain family interaction patterns, may contribute to the development of emotional lability and risk for self-injury. The authors explored the relation between peripheral serotonin levels and mother-child interaction patterns among typical (n = 21) and self-injuring (n = 20) adolescents. Findings revealed higher levels of negative affect and lower levels of both positive affect and cohesiveness among families of self-injuring participants. Peripheral serotonin was also correlated with the expression of positive affect within dyads. Furthermore, adolescents' serotonin levels interacted with negativity and conflict within dyads to explain 64% of the variance in self-injury. These findings underscore the importance of considering both biological and environmental risk factors in understanding and treating self-injuring adolescents. Keywordsself-injury; adolescent; serotonin; family; discussion Adolescent self-inflicted injury (SII), including suicide attempts and nonsuicidal self-harm, represents a significant public health concern. In addition to the emotional toll on adolescents and their families, adolescent SII costs the United States healthcare system over $150 million per year in inpatient hospitalization costs alone (Olfson, Gameroff, Marcus, Greenberg, & Despite being a leading cause of death among youths, suicide is a low base-rate phenomenon. Epidemiological data suggest that 1.6/100,000 youths ages 10-14 and 9.7/100,000 youths ages 14-19 die by suicide each year (U.S. Public Health Service, 1999). Although nonfatal selfinjury is more common than completed suicide, with as many as 8.8% of teens reporting a "suicide attempt" (Grunbaum et al., 2002), accurately predicting suicide and self-injury on the basis of known risk factors is currently not possible. Given the significance of this public health concern, specifying etiological factors that could improve our understanding of and our ability to prevent SII should become a national priority.Despite the urgency in furthering our understanding of SII, current data are quite limited. In all likelihood, future studies will identify etiological pathways to SII through detailed analyses of both biological vulnerabilities and environmental risk factors, as well as Biology × Environment interactions. Indeed, research suggests that many psychopathological outcomes are influenced by the interaction of biological and environmental influences (Kendler, Prescott, Myers, & Neale, 2003). Thus, etiological models of SII must address the complex interrelations among dynamic biological, psychological, and social systems. Although longitudi...