Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90-95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

On the variability of respiration in terrestrial ecosystems: moving beyond Q10

Respiration, which is the second most important carbon flux in ecosystems following gross primary productivity, is typically represented in biogeochemical models by simple temperature dependence equations. These equations were established in the 19th century and have been modified very little since then. Recent applications of these equations to data on soil respiration have produced highly variable apparent temperature sensitivities. This paper searches for reasons for this variability, ranging from biochemical reactions to ecosystem‐scale substrate supply. For a simple membrane‐bound enzymatic system that follows Michaelis–Menten kinetics, the temperature sensitivities of maximum enzyme activity (V_max) and the half‐saturation constant that reflects the affinity of the enzyme for the substrate (K_m) can cancel each other to produce no net temperature dependence of the enzyme. Alternatively, when diffusion of substrates covaries with temperature, then the combined temperature sensitivity can be higher than that of each individual process. We also present examples to show that soluble carbon substrate supply is likely to be important at scales ranging from transport across membranes, diffusion through soil water films, allocation to aboveground and belowground plant tissues, phenological patterns of carbon allocation and growth, and intersite differences in productivity. Robust models of soil respiration will require that the direct effects of substrate supply, temperature, and desiccation stress be separated from the indirect effects of temperature and soil water content on substrate diffusion and availability. We speculate that apparent Q₁₀ values of respiration that are significantly above about 2.5 probably indicate that some unidentified process of substrate supply is confounded with observed temperature variation.     

Rapid analysis of gene expression (RAGE) facilitates universal expression profiling.

Current techniques for analysis of gene expression either monitor one gene at a time, for example northern hybridization or RT-PCR methods, or are designed for the simultaneous analysis of thousands of genes, for example microarray hybridization or serial analysis of gene expression. To provide a flexible, intermediate scale alternative, a PCR-based method for the rapid analysis of gene expression has been developed which allows expression changes to be determined in either a directed search of known genes, or an undirected survey of unknown genes. A single set of reagents and reaction conditions allows analyses of most genes in any eukaryote. The method is useful for assaying on the order of tens to hundreds of genes in multiple samples. Control experiments indicate reliable detection of changes in gene expression 2-fold and greater, and sensitivity of detection better than 1 in 10 000. Analyses of over 400 genes in a mouse system transgenic for the E2F1 gene have identified several new downstream targets of E2F1, including Brca1 and Cdk7, in addition to several unidentified genes that are upregulated in the transgenic mice. Changes in expression of several genes related to apoptosis suggest a possible potentiation of apoptotic pathways in the transgenic keratinocytes.     

Large seasonal changes in Q10 of soil respiration in a beech forest

We analyzed one year of continuous soil respiration measurements to assess variations in the temperature sensitivity of soil respiration at a Danish beech forest. A single temperature function derived from all measurements across the year (Q₁₀ = 4.2) was adequate for estimating the total annual soil respiration and its seasonal evolution. However, Q₁₀'s derived from weekly datasets ranged between three in summer (at a mean soil temperature of 14 °C) and 23 in winter (at 2 °C), indicating that the annual temperature function underestimated the synoptic variations in soil respiration during winter. These results highlight that empirical models should be parameterized at a time resolution similar to that required by the output of the model. If the objective of the model is to simulate the total annual soil respiration rate, annual parameterization suffices. If however, soil respiration needs to be simulated over time periods from days to weeks, as is the case when soil respiration is compared to total ecosystem respiration during synoptic weather patterns, more short‐term parameterization is required. Despite the higher wintertime Q₁₀'s, the absolute response of soil respiration to temperature was smaller in winter than in summer. This is mainly because in absolute numbers, the temperature sensitivity of soil respiration depends not only on Q₁₀, but also on the rate of soil respiration, which is highly reduced in winter. Nonetheless, the Q₁₀ of soil respiration in winter was larger than can be explained by the decreasing respiration rate only. Because the seasonal changes in Q₁₀ were negatively correlated with temperature and positively correlated with soil moisture, they could also be related to changing temperature and/or soil moisture conditions.     

Climatic characteristics of heat waves and their simulation in plant experiments

Extreme events such as heat waves are emerging as a key facet of climate change, but to date, experiments on the impacts on plants are scarce. Experimental simulation of heat waves requires knowledge of regional heat wave characteristics, as plant responses depend heavily on meteorological conditions. We analysed nine Western European meteorological datasets, and found that heat waves occurring during the growing season in this region encompass more sunshine (+ 69%), lower precipitation (?78%) and a larger vapour pressure deficit (VPD) (+ 111%) compared with normal conditions. Possible consequences for plant responses are discussed, with emphasis on the likely seasonal variation of heat wave impacts. We explain why infrared heating (which typically increases VPD) is an appropriate technique for heat wave simulation. Finally, we advocate experiments to take into account the smaller nighttime compared with daytime temperature increases observed during heat waves, and the precipitation deficits before and during heat waves.     

Impact of habitat fragmentation on the genetics of populations in dendritic landscapes

1. The effect of habitat fragmentation on freshwater species has been addressed using brown trout Salmo trutta L. as a model species with a dendritic population structure. 2. Microsatellite loci were employed as molecular markers. Levels of gene flow and population subdivision were determined in more than 1200 brown trout individuals inhabiting four south European rivers with contrasting patterns of fragmentation, defined by the presence of barriers. 3. The genetic units in the four rivers were restricted by artificial barriers, and gene flow among samples within each river was associated with the level of fragmentation of the river. 4. Loss of genetic diversity and dislocation of the dendritic model have been detected in fragmented rivers. These results emphasise the importance of mitigating the impact of dams by constructing passages to restore gene flow along the river, for fish and other migratory species, as well as the need for caution in relation to stocking in isolated areas to avoid problems of inbreeding.     

Household Transmission of Influenza A(H1N1)pdm09 in the Pandemic and Post-Pandemic Seasons

Background

The transmission of influenza viruses occurs person to person and is facilitated by contacts within enclosed environments such as households. The aim of this study was to evaluate secondary attack rates and factors associated with household transmission of laboratory-confirmed influenza A(H1N1)pdm09 in the pandemic and post-pandemic seasons.

Methods

During the 2009–2010 and 2010–2011 influenza seasons, 76 sentinel physicians in Navarra, Spain, took nasopharyngeal and pharyngeal swabs from patients diagnosed with influenza-like illness. A trained nurse telephoned households of those patients who were laboratory-confirmed for influenza A(H1N1)pdm09 to ask about the symptoms, risk factors and vaccination status of each household member.

Results

In the 405 households with a patient laboratory-confirmed for influenza A(H1N1)pdm09, 977 susceptible contacts were identified; 16% of them (95% CI 14–19%) presented influenza-like illness and were considered as secondary cases. The secondary attack rate was 14% in 2009–2010 and 19% in the 2010–2011 season (p = 0.049), an increase that mainly affected persons with major chronic conditions. In the multivariate logistic regression analysis, the risk of being a secondary case was higher in the 2010–2011 season than in the 2009–2010 season (adjusted odds ratio: 1.72; 95% CI 1.17–2.54), and in children under 5 years, with a decreasing risk in older contacts. Influenza vaccination was associated with lesser incidence of influenza-like illness near to statistical significance (adjusted odds ratio: 0.29; 95% CI 0.08–1.03).

Conclusion

The secondary attack rate in households was higher in the second season than in the first pandemic season. Children had a greater risk of infection. Preventive measures should be maintained in the second pandemic season, especially in high-risk persons.