Effect of simulated tillage on microbial autotrophic CO2 fixation in paddy and upland soils.

Tillage is a common agricultural practice affecting soil structure and biogeochemistry. To evaluate how tillage affects soil microbial CO2 fixation, we incubated and continuously labelled samples from two paddy soils and two upland soils subjected to simulated conventional tillage (CT) and no-tillage (NT) treatments. Results showed that CO2 fixation ((14)C-SOC) in CT soils was significantly higher than in NT soils. We also observed a significant, soil type- and depth-dependent effect of tillage on the incorporation rates of labelled C to the labile carbon pool. Concentrations of labelled C in the carbon pool significantly decreased with soil depth, irrespective of tillage. Additionally, quantitative PCR assays revealed that for most soils, total bacteria and cbbL-carrying bacteria were less abundant in CT versus NT treatments, and tended to decrease in abundance with increasing depth. However, specific CO2 fixation activity was significantly higher in CT than in NT soils, suggesting that the abundance of cbbL-containing bacteria may not always reflect their functional activity. This study highlights the positive effect of tillage on soil microbial CO2 fixation, and the results can be readily applied to the development of sustainable agricultural management.

The heritable basis and cost of colour plasticity in coastrange sculpins.

Both genetic and plastic traits contribute to adaptation in novel environments. Phenotypic plasticity can facilitate adaptation by allowing for existence in a wider range of conditions and a faster response to environmental change than gene-based selection. Coastrange sculpins (Cottus aleuticus) colonize new and variable streams arising in the wake of receding glaciers in south-east Alaska, and substrate-matching plasticity may enhance colonization success by reducing detection by visual predators. As part of a long-term study of the fitness consequences of colour plasticity and its capacity to respond to both positive and negative selection, we investigated whether it is heritable and costly. We raised full-sib broods of sculpins in the laboratory: one half of each brood was raised in white containers, the other half in black. After 4 months, we digitally analysed their colour and found significant but weak heritability in both baseline colour and colour plasticity. To investigate the cost of colour plasticity, we compared the growth and mortality rates of juvenile sculpins reared under constant substrate colours to those reared on substrates that changed colour frequently, and compared growth rates among sculpin that differed in their colour change ability. We found evidence of small costs of plasticity, consistent with other studies of natural populations. Evidence of heritable genetic variation for plasticity and small costs to its maintenance and expression contributes to explanations of how plasticity is variable and persistent among wild populations and underscores its ability to respond both positively and negatively to selection in variable habitats.

Pervasive sex-linked effects on transcription regulation as revealed by expression quantitative trait loci mapping in lake whitefish species pairs (Coregonus sp., Salmonidae).

Mapping of expression quantitative trait loci (eQTL) is a powerful means for elucidating the genetic architecture of gene regulation. Yet, eQTL mapping has not been applied toward investigating the regulation architecture of genes involved in the process of population divergence, ultimately leading to speciation events. Here, we conducted an eQTL mapping experiment to compare the genetic architecture of transcript regulation in adaptive traits, differentiating the recently evolved limnetic (dwarf) and benthic (normal) species pairs of lake whitefish. The eQTL were mapped in three data sets derived from an F(1) hybrid-dwarf backcrossed family: the entire set of 66 genotyped individuals and the two sexes treated separately. We identified strikingly more eQTL in the female data set (174), compared to both male (54) and combined (33) data sets. The majority of these genes were not differentially expressed between male and female progeny of the backcross family, thus providing evidence for a strong pleiotropic sex-linked effect in transcriptomic regulation. The subtelomeric region of a linkage group segregating in females encompassed >50% of all eQTL, which exhibited the most pronounced additive effects. We also conducted a direct comparison of transcriptomic profiles between pure dwarf and normal progeny reared in controlled conditions. We detected 34 differentially expressed transcripts associated with eQTL segregating only in sex-specific data sets and mostly belonging to functional groups that differentiate dwarf and normal whitefish in natural populations. Therefore, these eQTL are not related to interindividual variation, but instead to the adaptive and historical genetic divergence between dwarf and normal whitefish. This study exemplifies how the integration of genetic and transcriptomic data offers a strong means for dissecting the functional genomic response to selection by separating mapping family-specific effects from genetic factors under selection, potentially involved in the phenotypic divergence of natural populations.

Microbial analysis of soil and groundwater from a gasworks site and comparison with a sequenced biological reactive barrier remediation process.

AIMS: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. METHODS AND RESULTS: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. CONCLUSION: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. SIGNIFICANCE AND IMPACT OF THE STUDY: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.

Salivary nitrate--an ecological factor in reducing oral acidity.

Human oral cavities represent a novel environment with a constant supply of concentrated nitrate. For humans, over 80% of dietary nitrate originates from fruit and vegetables. With a healthy, balanced diet, rich in fruit and vegetables, the concentration of nitrate in saliva can reach up to more than three times the European drinking water standard. The physiological function of the active excretion of salivary nitrate is unknown. Furthermore, little is known of the ecological function of oral nitrate and the effect on the oral environment during its subsequent oral microbial conversions. The objectives of the research were to investigate the effect on salivary pH coupled with oral microbial nitrate and/or nitrite reduction. Human saliva samples were incubated anaerobically in the presence of 111.0 mmol glucose (2%), with and without 1.5 mmol nitrate/nitrite, and pH and nitrate/nitrite consumption were measured during the time-course of the incubations. We found that anaerobic incubation of saliva containing a mixture of oral bacteria in the presence of nitrate/nitrite substrates and glucose resulted in a higher pH than was found in controls in the absence of nitrate/nitrite. These results suggest that the presence of these electron acceptors repressed acid fermentation, or increased alkali production, or consumed acid produced, thus reducing salivary acidity. This finding identifies salivary nitrate as a possible ecological factor in reducing oral acidity. The possibility that a symbiotic relationship between host nitrate excretion and nitrate-reducing microorganisms might help to protect against tooth decay should be explored further.

Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway.

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.

Genome-wide epistatic interaction analysis reveals complex genetic determinants of circadian behavior in mice.

Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F(2) hybrid mice. We have characterized variation in this panel of F(2) mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.

Ecological and physiological analyses of Pseudomonad species within a phenol remediation system.

A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy.

Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition.

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

Long term azathioprine fails to prevent onset of multiple sclerosis: report of two cases.

Azathioprine is an immunosuppressive drug widely used in the treatment of chronic inflammatory diseases, including Multiple Sclerosis (MS). We report two patients who developed the first manifestations of clinically definite multiple sclerosis while on long term (3.5 and 10 years, respectively) treatment with azathioprine for Crohn's disease. Both patients developed the first MS symptoms during a quiescent phase of their inflammatory bowel disease. These cases show that long term azathioprine, while possibly maintaining inflammatory bowel disease under control, could not prevent the onset of MS. Multiple Sclerosis (2000) 6 362 - 363

Bacterial community structure and physiological state within an industrial phenol bioremediation system.

The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the alpha, beta and gamma subclasses of the class Proteobacteria [alpha-, beta-, and gamma-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the gamma-Proteobacteria. The alpha subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the beta subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The gamma subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the gamma-Proteobacteria and the Cytophaga-Flavobacteria during this bioremediation process was highlighted.

Density gradient separation of active and non-active cells from natural environments.

We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities. The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates. The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation. The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples. Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate. Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.

The Xenopus clock gene is constitutively expressed in retinal photoreceptors.

Many aspects of normal retinal physiology are controlled by a retinal circadian clock. In Xenopus laevis, the photoreceptor cells within the retina contain a circadian clock that controls melatonin release. In this report we present the cloning and characterization of the Xenopus homolog of the Clock gene, known to be critical for normal circadian behavioral rhythms in the mouse. The Xenopus Clock gene is expressed primarily in photoreceptors within the eye and is expressed at constant levels throughout the day. Analysis of other tissues revealed that, as in other species, the Xenopus Clock gene is widely expressed. This characterization of the Clock gene provides a useful tool for further exploration of the role of the circadian clock in normal retinal function.

Effects of targeting herpes simplex virus type 1 gD to the endoplasmic reticulum and trans-Golgi network.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was modified to encode targeting signals known to localize proteins to either the endoplasmic reticulum (ER) or the trans-Golgi network. These motifs conferred the predicted targeting properties on gD in transfected cells as judged by immunofluorescence staining, and the exclusion of targeted gD from the cell surface was confirmed by the fact that these molecules exhibited substantially reduced activity in cell-cell fusion assays. Recombinant viruses expressing Golgi-targeted forms of gD grew to wild-type levels in noncomplementing cells, exhibited unaltered particle/infectivity ratios, and were found to contain wild-type levels of gD, whereas a recombinant expressing ER-retained gD was helper cell dependent and, when grown on noncomplementing cells, produced virions of low specific infectivity with greatly reduced levels of gD. These data imply that HSV-1 acquires its final membrane from a post-ER compartment and lend support to the view that the virus undergoes de-envelopment and reenvelopment steps during virus egress.

Two-surgeon approach to thoracic outlet syndrome: long-term outcome.

An orthopaedic surgeon and a vascular surgeon jointly conducted 30 operations for thoracic outlet syndrome in 27 patients, having done the preoperative assessments in conjunction with a neurologist. Anterior scalenectomy was performed by the supraclavicular route except in one case where the infraclavicular route was used. The further surgical procedure was tailored to the abnormalities identified--i.e. resection of cervical rib or band, or medial scalenectomy. The first rib was spared. At median follow-up of 37 months (range 3-228) results were judged excellent or good on 26/30 sides (87%); on the three occasions when scalenectomy alone was performed, the results were only fair or poor. There were no major complications and no patient required reoperation. The long-term outcome in this series suggests that, with multidisciplinary assessment and two-surgeon operative treatment, good results can be obtained by the supraclavicular route without resection of the first rib.

Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription.

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.

Automated measurement of mouse freezing behavior and its use for quantitative trait locus analysis of contextual fear conditioning in (BALB/cJ x C57BL/6J)F2 mice.

The most commonly measured mouse behavior in fear conditioning tests is freezing. A technical limitation, particularly for genetic studies, is the method of direct observation used for quantifying this response, with the potential for bias or inconsistencies. We report the use of a computerized method based on latency between photobeam interruption measures as a reliable scoring criterion in mice. The different computer measures obtained during contextual fear conditioning tests showed high correlations with hand-scored freezing; r values ranged from 0.87 to 0.94. Previously reported strain differences between C57BL/6J and DBA/2J in context-dependent fear conditioning were also detected by the computer-based system. In addition, the use of computer-scored freezing of 199 (BALB/cJ x C57BL/6J)F2 mice enabled us to detect a suggestive gender-dependent chromosomal locus for contextual fear conditioning on distal chromosome 8 by QTL analysis. Automation of freeze scoring would significantly increase efficiency and reliability of this learning and memory test.

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.