Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

In vitro colony formation by cryopreserved primary human tumor cells

The effect of cryopreservation on the ability of primary human cancer cells to form colonies in a two-layer agar system was examined. Although considerable variation occurred, concentrations of 5 or 10% dimethylsulfoxide employed with slow freezing rates allowed survival of colonies in the range 20-40% or greater of nonfrozen controls. The methods used in this study do not require elaborate freezing equipment, and can be used for the cryopreservation of a wide variety of types of cancers.     

Amphibian chytrid fungus and ranaviruses in the Northwest Territories, Canada

Pathogens can cause serious declines in host species, and knowing where pathogens associated with host declines occur facilitates understanding host-pathogen ecology. Suspected drivers of global amphibian declines include infectious diseases, with 2 pathogens in particular, Batrachochytrium dendrobatidis (Bd) and ranaviruses, causing concern. We explored the host range and geographic distribution of Bd and ranaviruses in the Taiga Plains ecoregion of the Northwest Territories, Canada, in 2007 and 2008. Both pathogens were detected, greatly extending their known geographic distributions. Ranaviruses were widespread geographically, but found only in wood frogs. In contrast, Bd was found at a single site, but was detected in all 3 species of amphibians in the survey area (wood frogs, boreal chorus frogs, western toads). The presence of Bd in the Northwest Territories is not congruent with predicted distributions based on niche models, even though findings from other studies at northern latitudes are consistent with those same models. Unexpectedly, we also found evidence that swabs routinely used to collect samples for Bd screening detected fewer infections than toe clips. Our use and handling of the swabs was consistent with other studies, and the cause of the apparent lack of integrity of swabs is unknown. The ranaviruses detected in our study were confirmed to be Frog Virus 3 by sequence analysis of a diagnostic 500 bp region of the major capsid protein gene. It is unknown whether Bd or ranaviruses are recent arrivals to the Canadian north. However, the genetic analyses required to answer that question can inform larger debates about the origin of Bd in North America as well as the potential effects of climate change and industrial development on the distributions of these important amphibian pathogens.     

Thermal chip fabrication with arrays of sensors and heaters for micro-scale impingement cooling heat transfer analysis and measurements

The design and fabrication for a thermal chip with an array of temperature sensors and heaters for study of micro-jet impingement cooling heat transfer process are presented. This thermal chip can minimize the heat loss from the system to the ambient and provide a uniform heat flux along the wall, thus local heat transfer processes along the wall can be measured and obtained. The fabrication procedure presented can reach a chip yield of 100%, and every one of the sensors and heaters on the chip is in good condition. In addition, micro-jet impingement cooling experiments are performed to obtain the micro-scale local heat transfer Nusselt number along the wall. Flow visualization for the micro-impinging jet is also made. The experimental results indicate that both the micro-scale impinging jet flow structure and the heat transfer process along the wall is significantly different from the case of large-scale jet impingement cooling process.     

Evaluation of ultrasound velocity measurements for estimating protease activities using casein as substrate

Ultrasonic resonator technology (URT) was compared with the well established UV–Vis/ninhydrin assay to estimate protease activities in defined buffer systems. Hydrolysis of casein was measured using subtilisin, trypsin, halophilic protease from Haloferax mediterranei and Bacillus lentus alkaline protease. Sensitivity, reproducibility, working range as well as the limit of detection and the limit of quantification were comparable for both methods. Salt concentrations (0.5 M NaCl) interfered with the URT method. The quantification of protease activity by URT was possible when the product concentration measured by the UV–Vis/ninhydrin assay was correlated to the corresponding ultrasonic velocity signals.     

Design and fabrication development of a micro flow heated channel with measurements of the inside micro-scale flow and heat transfer process

The current work provides a design and fabrication technique for a micro channel system that can provide a uniform heat flux boundary condition on the channel wall and a well insulation on the wall to prevent heat loss from the channel to the outside ambient. Therefore, detailed micro-scale flow and heat transfer process and information along the channel can be studied. Semiconductor sensor material was selected to fabricate both the heaters and the arrays of temperature sensors on a silicon substrate. These heaters and sensors were then moved to a low thermal conductivity epoxy-glass substrate for fabrication of the channel. Design consideration and fabrication techniques involved in this processes will be discussed. A final measurement for the validation of the heaters and the sensors fabricated and a study of the flow friction behavior and the heat transfer coefficient distributions inside the micro channel will be presented. The local Nusselt number distrubution inside the micro channel is reported the first time in the open literature.     

Stability of the I-motif structure is related to the interactions between phosphodiester backbones

The i-motif DNA tetrameric structure is formed of two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. The four phosphodiester backbones forming the structure define two narrow and wide grooves. The short interphosphate distances across the narrow groove induce a strong repulsion which should destabilize the tetramer. To investigate this point, molecular dynamics simulations were run on the [d(C2)]4 and [d(C4)]4 tetramers in 3'E and 5'E topologies, for which the interaction of the phosphodiester backbones through the narrow groove is different. The analysis of the simulations, using the Molecular Mechanics Generalized Born Solvation Area and Molecular Mechanics Poisson-Boltzmann Solvation Area approaches, shows that it is the van der Waals energy contribution which displays the largest relative difference between the two topologies. The comparison of the solvent-accessible area of each topology reveals that the sugar-sugar interactions account for the greater stability of the 3'E topology. This stresses the importance of the sugar-sugar contacts across the narrow groove which, enforcing the optimal backbone twisting, are essential to the base stacking and the i-motif stability. Tighter interactions between the sugars are observed in the case of N-type sugar puckers.