Real-time monitoring of rolling-circle amplification using a modified molecular beacon design

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2′-O-Me-RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.     

DNA fragmentation and morphological changes in apoptotic human lymphocytes

Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that >/=50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density.The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.     

Individual differences in sensitivity to the odor of 4,16-androstadien-3-one

Individual differences in sensitivity to the putative human pheromone androstadienone were investigated in three experiments. In experiment 1, the absolute detection threshold for androstadienone was determined to be 211 micro M using the method of constant stimuli. Detection for the related compound estratetraenol was also investigated but a threshold could not be determined. In experiment 2, using an adaptive threshold test on 100 participants, the sensitivity distribution for androstadienone, but not for the reference odor phenylethyl alcohol, was bimodal, with a smaller group of individuals with a high sensitivity to androstadienone (supersmellers). A lack of correlation between thresholds for androstadienone and phenylethyl alcohol further suggested that the bimodality for androstadienone was not due to individuals with a high general olfactory sensitivity. In line with an earlier observation, there was a statistical tendency for women to be more sensitive to androstadienone than men. Results of experiment 3 preclude the possibility that the bimodal sensitivity distribution for androstadienone would depend on individual differences in trigeminal activation. Altogether, the current study suggests that olfactory sensitivity to androstadienone is bimodally distributed in the population with a subgroup consisting of highly sensitive people.     

Medroxyprogesterone priming and response to the ram effect in Corriedale ewes during the nonbreeding season

The "ram effect" (RE) is an inexpensive technique that allows farmers to obtain out-of-season lambs. Five hundred and ninety-six Corriedale ewes were used in three experiments to determine the effectiveness of different medroxyprogesterone (MAP) treatments associated with the ram effect during the nonbreeding season. The aim of the first experiment was to evaluate the effectiveness of short-term (6-day) MAP priming. We obtained similar results in estrus incidence and fertility after using MAP sponges for 6, 9, and 13 days. In the second experiment, we compared the effect of sponges containing 20, 40, or 60 mg of MAP used in 6-day priming. Estrous behavior and fertility were not affected by dosage. In the third experiment, 2.5mg of MAP was administered in single treatments 0, 1, 3, or 5 days before the introduction of the rams. Medroxyprogesterone administration 1, 3, or 5 days before the introduction of the rams concentrated estrus in ewes 17 to 20 days later.     

Survival and growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

Prenatal sex ratios influence sexual dimorphism in a reptile

The prenatal environment influences offspring traits in a variety of ways and in a wide range of taxa. For example, maternal allocation of steroids to the eggs influences offspring traits in birds, and in some mammals the intrauterine position influences morphological, behavioural, and physiological traits due to sex-related steroid transfer between sibling fetuses. We show that similar phenomena occur in the common lizard (Lacerta vivipara), a viviparous reptile. Females developing in male-biased clutches had a more masculine allometry (relatively larger heads) at parturition than females developing in female-biased clutches. Males were correspondingly feminized in female-biased clutches. The effects could either be due to diffusion of steroids produced by the offspring or by a general tendency for females to allocate steroids according to the sex ratio of her clutch. Subsequent to parturition, the sexes differed in their growth trajectories depending on sex ratio environment. In males, the difference in allometry between sex ratio environments remained over time, whereas in females the corresponding effect disappeared.     

Techno-economic evaluation of producing ethanol from softwood: comparison of SSF and SHF and identification of bottlenecks

The aim of the study was to evaluate, from a technical and economic standpoint, the enzymatic processes involved in the production of fuel ethanol from softwood. Two base case configurations, one based on simultaneous saccharification and fermentation (SSF) and one based on separate hydrolysis and fermentation (SHF), were evaluated and compared. The process conditions selected were based mainly on laboratory data, and the processes were simulated by use of Aspen plus. The capital costs were estimated using the Icarus Process Evaluator. The ethanol production costs for the SSF and SHF base cases were 4.81 and 5.32 SEK/L or 0.57 and 0.63 USD/L (1 USD = 8.5SEK), respectively. The main reason for SSF being lower was that the capital cost was lower and the overall ethanol yield was higher. A major drawback of the SSF process is the problem with recirculation of yeast following the SSF step. Major economic improvements in both SSF and SHF could be achieved by increasing the income from the solid fuel coproduct. This is done by lowering the energy consumption in the process through running the enzymatic hydrolysis or the SSF step at a higher substrate concentration and by recycling the process streams. Running SSF with use of 8% rather than 5% nonsoluble solid material would result in a 19% decrease in production cost. If after distillation 60% of the stillage stream was recycled back to the SSF step, the production cost would be reduced by 14%. The cumulative effect of these various improvements was found to result in a production cost of 3.58 SEK/L (0.42 USD/L) for the SSF process.     

Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Effects of sex, body size, temperature, and location on the antipredator tactics of free-ranging gartersnakes (Thamnophis sirtalis, Colubridae)

Gartersnakes (Thamnophis sirtalis parietalis) in southern Manitoba are subject to intense predation (primarily by crows) duringtheir spring breeding season. The huge numbers of snakes providea unique opportunity to quantify behavioral traits. We simulatedpredator attacks by "pecking" more than 500 free-ranging snakes,to explore the determinants of snake response. Snakes respondedto a human finger in the same way as they did to a more realisticstimulus (a model crow). A snake's response to attack dependedon several factors, which interacted in complex ways. The primaryinfluences on response were body temperature (warmer snakes tended to flee, whereas colder snakes remained cryptic or flattenedand/or gaped and struck) and sex (males were more likely toflee). Responses also depended on microhabitat (i.e., insidethe winter den versus in adjacent grassland) and on the snake'sprior activity (e.g., courting snakes often ignored our closeapproach). These factors interacted in significant ways; for example, snakes outside the den were smaller and warmer thanthose inside, male snakes were smaller and warmer than females,and mean body temperatures were higher in larger snakes withineach sex. Thus, a snake's body size and its location affectedits defensive response indirectly (via their influence on bodytemperature). Our results differ from those of previous studiesand suggest that antipredator responses in these animals dependin a flexible and complex way upon biotic and abiotic variables.Interactions among these variables also must be consideredbefore we can identify underlying causal processes.