Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Distinguishing kinds of prior dominance and subordination experiences in males of green swordtail fish (Xiphophorus helleri)

In experiments, there are usually two general ways of obtaining dominants and subordinates to test for the effect of recent experience upon ulterior behavior and dominance. One is to ‘impose’ such an experience on the contestants by a priori deciding which individual of the pair will become the dominant and which will become the subordinate through the use of rigged contests. The second technique is to let contestants ‘self-select’ the winner and loser by waiting for the spontaneous outcome of dyadic encounters between two usually well matched opponents. These two techniques of obtaining dominants and subordinates probably represent extreme cases on a single continuum of investment made by animals to settle dominance. To test this, we compared dominants and subordinates obtained from these two techniques in Xiphophorus fish males. It was found that pairs obtained through rigged contest (R) were much more aggressive in subsequent encounters than pairs in which the dominant and subordinate could self-select (S). They recuperated more rapidly from handling, initiated contact earlier, took more time to assess each other, and fought for a longer period of time. Prior-winners and prior-losers of the R condition more frequently relied on aggressive behavior during contest than that of the S condition. As a consequence, prior-winners and prior-losers of the R condition won equally the subsequent contest. On the contrary, prior-winners of the S condition defeated their prior-loser opponent in a majority of cases. These results can be tentatively explained by the following principle, winning or losing against a well matched opponent would leave more ‘experience’ than winning over a much weaker opponent, or losing to a much stronger one. This reinforces the hypothesis that prior-experiences are not qualitative states but come in various degrees.     

Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

Photosynthetic responses of Eucalyptus nitens (Deane and Maiden) Maiden to green pruning

 Three-year-old Eucalyptus nitens (Deane and Maiden) Maiden trees and 1-year-old ramets of a single clone of E. nitens were pruned to remove 0, 50% or 70% of the green crown length. This was equivalent to removal of 0, 55% or 88% of foliage area of trees, and 0, 77% or 94% of foliage area of ramets. CO₂ assimilation (A) and stomatal conductance (g_s) were measured at constant illumination in five height zones and three foliage-age classes of trees over a 16-month period following pruning. Foliar nitrogen (N) and phosphorus (P) concentrations were determined for each measurement time during the first 12 months of the experiment. In ramets A and g_s were measured in four height zones and two foliage-age classes over a six-week period, and N and P concentrations were measured only once, at the end of the experiment. Rates of A increased by up to 175% following pruning. This response occurred throughout the canopy irrespective of position in the crown or foliage age. The magnitude of the response was generally greater in ramets than in trees, and increased with increasing severity of pruning. The initiation of the response was later, and the duration of the response was longer, in trees than ramets. In the lower crown of trees there was evidence of delayed senescence following pruning. Photosynthetic enhancement was not related to changes in foliar N concentrations. The ratio of A/N increased in many zones following pruning, especially after more severe defoliation. There was no evidence that changes in P concentrations were responsible for the result. The increases in A may have been related to changes in g_s, as maximum values of g_s were greater, and the ratio of A/g_s was generally lower, in pruned than unpruned plants. Received: 31 December 1996 / Accepted: 19 August 1997     

Prediction and interpretation of the antioxidant capacity of green tea from dissimilar chromatographic fingerprints

Previously, multivariate calibration techniques have been successfully applied to model and predict the antioxidant activity of green tea from its chromatographic fingerprint. Since the selectivity differences between dissimilar chromatographic systems have already been valuably used in several applications, in this paper it is studied whether combining the complementary information contained in two dissimilar fingerprints can improve the predictive capacity of the multivariate calibration model. The simplest way of combining the data is concatenating both fingerprints for each sample. The resulting matrix can then be subjected to Orthogonal Projections to Latent Structures (O-PLS). Unfortunately, this approach resulted in a more complex model with a prediction error of about the average of the errors obtained with the individual fingerprints. Secondly, only the peaks with high loading and low orthogonal loading from both chromatograms were included in the O-PLS model. This resulted in a reduced complexity, but not in better predictions, probably due to a lack of complementarity of the information concerning the antioxidant capacity. Finally, the concatenated fingerprints were subjected to stepwise multiple linear regression (MLR) in order to build a model based on the variables most correlated with the antioxidant capacity. The obtained prediction error was lower than those of both previous approaches, but still higher than the error of the model based on a single analysis. This is probably again caused by a lack of complementarity in the variables. Nevertheless, it was advantageous to develop fingerprints on dissimilar system, because it enables to choose the most suited chromatographic profile to build a multivariate calibration model for the considered purpose. In contrast to what was expected, the study showed that the most simple (so the worst separated) fingerprints resulted in the best predictions. On the other hand, a more complex fingerprint in which more compounds are separated is still important to improve the interpretability of the model.     

Limnology of the Green Lakes Valley: phytoplankton ecology and dissolved organic matter biogeochemistry at a long-term ecological research site

ABSTRACT

Background: Surface waters are the lowest points in the landscape, and therefore serve as excellent integrators and indicators of changes taking place in the surrounding terrestrial and atmospheric environment.

Aims: Here we synthesise the findings of limnological studies conducted during the past 15 years in streams and lakes in the Green Lakes Valley, which is part of the Niwot Ridge Long Term Ecological Research (LTER) Site.

Methods: The importance of these studies is discussed in the context of aquatic ecosystems as indicators, integrators, and regulators of environmental change. Specifically, investigations into climatic, hydrologic, and nutrient controls on present-day phytoplankton, and historical diatom, community composition in the alpine lake, Green Lake 4, are reviewed. In addition, studies of spatial and temporal patterns in dissolved organic matter (DOM) biogeochemistry and reactive transport modelling that have taken place in the Green Lakes Valley are highlighted.

Results and conclusions: The findings of these studies identify specific shifts in algal community composition and DOM biogeochemistry that are indicative of changing environmental conditions and provide a framework for detecting future environmental change in the Green Lakes Valley and in other alpine watersheds. Moreover, the studies summarised here demonstrate the importance of long-term monitoring programmes such as the LTER programme.     

Fetal vs adult mesenchymal stem cells achieve greater gene expression,but less osteoinduction

AIM: To investigate adenoviral transduction in mesenchymal stem cells(MSCs) and effects on stemness in vitro and function as a cell therapy in vivo.METHODS: Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine(PEI)-mediated transfection of pc DNA3-e GFP or adenoviral transduction of green fluorescent protein(GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness(i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2(BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology.RESULTS: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs(81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs(78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs(7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression(0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs(1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitrowere not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult(P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells(P < 0.05). AdBMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs(83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone(1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05).CONCLUSION: Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.     

Squeezed flow preconcentration for probe tip biosensors

The preconcentration of analytes improves sensing using probe tips. In this work, we report a method based on creating a squeeze flow between a cylinder and circular coverslip to preconcentrate material at the liquid–gas interface while allowing a probe tip to be readily inserted there. In verification tests using enhanced green fluorescent protein, this capacity is proven. We estimated a 9.7 times increase in probability for fluorophores to be picked up at the tip using inference from fluorescence intensity distributions found. The method is expeditious, simple, and inexpensive, and it does not require any electrical energy source to operate.     

绿海龟稚龟性别鉴定及临界温度

通过组织形态学鉴定、羊水和血液中性激素测定等手段,对惠东港口海龟保护区不同恒温下孵化的绿海龟(Chelonia mydas)进行研究,寻求绿海龟稚龟性别鉴定的最佳方法。结果显示,(1)组织形态学,绿海龟性腺切片分皮层和髓质两部分,卵巢的皮层较厚、基质中有大小不一的未成熟卵细胞,髓质中空腔较少;睾丸的皮层较薄,髓质中空腔较多,呈现出显著的被基质包围的髓质索。(2)性激素测定,当雌二醇与睾酮含量之比E2∶T1.5时,孵出的稚龟为雄性(♂);当E2∶T1.5时,为雌性(♀)。(3)绿海龟性别决定的临界温度在29.4～29.5℃之间,此时孵出稚龟的性比约为1∶1。本文还讨论了各种不同性别鉴定方法的优劣和时期选择。