A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 10² – 4.39 × 10³ s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8–5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.     

Reproduction site preference and performance by sexuparae,and mating behavior of their sexual generation on the primary host plant in a heteroecious aphid,Neothoracaphis yanonis (Homoptera: Aphididae)

We studied reproduction site preference and performance by the sexuparae (autumnal migrants) of Neothoracaphis yanonis and mating behavior of their sexual generation on its primary host plant, Distylium racemosum. The sexuparae preferred younger leaves of D. racemosum for settlement and imbibing leaf sap, and they produced more offspring there than on older leaves. Thus, it is suggested that the sexuparae selected more nutritious younger leaves to increase their own fecundity. The offspring consist of yellowish dark-grey and creamy yellow type nymphs, which develop into small males and large oviparae, respectively. Yellowish dark-grey nymphs were deposited gregariously on the basal part of the abaxial surface of a leaf blade, while creamy yellow nymphs were deposited evenly over the abaxial surface. Such a localized distribution pattern probably resulted from sexuparae's strategy to enhance a possibility for their male offspring to mate with oviparae at the base of leaf blade, over which oviparae crawl to twigs. During mating and sometimes afterwards, the ovipara incidentally carried a male on her back and crawled downward on twig to find an axillary bud for oviposition. This behavior may be advantageous to males, who can guard their mate during this period against other conspecific males. Such mate-guarding behavior seems to be related to the development of dual mate-seeking strategies, in which males try to copulate, first at the basal part of midrib, and second on the axillary bud.     

Neural systems for preparatory control of imitation

Humans have an automatic tendency to imitate others. Previous studies on how we control these tendencies have focused on reactive mechanisms, where inhibition of imitation is implemented after seeing an action. This work suggests that reactive control of imitation draws on at least partially specialized mechanisms. Here, we examine preparatory imitation control, where advance information allows control processes to be employed before an action is observed. Drawing on dual route models from the spatial compatibility literature, we compare control processes using biological and non-biological stimuli to determine whether preparatory imitation control recruits specialized neural systems that are similar to those observed in reactive imitation control. Results indicate that preparatory control involves anterior prefrontal, dorsolateral prefrontal, posterior parietal and early visual cortices regardless of whether automatic responses are evoked by biological (imitative) or non-biological stimuli. These results indicate both that preparatory control of imitation uses general mechanisms, and that preparatory control of imitation draws on different neural systems from reactive imitation control. Based on the regions involved, we hypothesize that preparatory control is implemented through top-down attentional biasing of visual processing.     

Efficient species identification of pine marten (<Emphasis Type="Italic">Martes martes</Emphasis>) and red fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) scats using a 5′ nuclease real-time PCR assay

Monitoring wildlife species by DNA identification of samples collected non-invasively is an important tool in conservation management. DNA identification of species from faecal (scat) samples is problematic due to the small quantities and poor quality of the DNA isolated from such samples. This study demonstrates the use of real-time PCR technology in the identification of red fox (Vulpes vulpes) and pine marten (Martes martes). It is shown that real-time PCR can be used to identify fox and pine marten by either melting curve analysis (Tm determination) with SYBR Green 1 detection or by the use of species specific fluorogenic probes. The technique is shown to work efficiently with scat DNA.     

Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization

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Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid

An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD⁺. Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD⁺ regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD⁺ as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA‐pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25°C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009