Force amplification response of actin filaments under confined compression

Actin protein is a major component of the cell cytoskeleton, and its ability to respond to external forces and generate propulsive forces through the polymerization of filaments is central to many cellular processes. The mechanisms governing actin's abilities are still not fully understood because of the difficulty in observing these processes at a molecular level. Here, we describe a technique for studying actin–surface interactions by using a surface forces apparatus that is able to directly visualize and quantify the collective forces generated when layers of noninterconnected, end-tethered actin filaments are confined between 2 (mica) surfaces. We also identify a force-response mechanism in which filaments not only stiffen under compression, which increases the bending modulus, but more importantly generates opposing forces that are larger than the compressive force. This elastic stiffening mechanism appears to require the presence of confining surfaces, enabling actin filaments to both sense and respond to compressive forces without additional mediating proteins, providing insight into the potential role compressive forces play in many actin and other motor protein-based phenomena.

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176-bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176-bp target compared with a larger 288-bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR-based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).

Integrated DNA extraction and amplification using electrokinetic pumping in a microfluidic device

An integrated system employing anion exchange for the extraction of DNA from biological samples prior to polymerase chain reaction DNA amplification has been developed, based on microfluidic methodology utilising electrokinetic pumping. In this system, the biological samples were added directly to chitosan-coated silica beads to facilitate DNA immobilisation. The purified, pre-concentrated DNA was then eluted using a combination of electro-osmotic flow enhanced with electrophoretic mobility, which enable DNA to be transported by both mechanisms into the DNA amplification chamber. Through optimisation of the DNA elution conditions, average DNA extraction efficiencies of 69.1% were achievable. Subsequent DNA amplification performed on the microfluidic system demonstrated not only the ability to use electrokinetic movement to integrate the two processes on a single device, but also that the quality and quantity of DNA eluted was suitable for downstream analysis. This work offers an attractive real-world to chip interface and a route to simpler Lab-on-a-Chip technology which eliminates the need for moving parts.

Validation of a multiplex reverse transcription and pre-amplification method using TaqMan(®) MicroRNA assays.

Since the discovery of microRNAs (miRNAs), different approaches have been developed to label, amplify and quantify miRNAs. The TaqMan(®) technology, provided by Applied Biosystems (ABIs), uses a stem-loop reverse transcription primer system to reverse transcribe the RNA and amplify the cDNA. This method is widely used to identify global differences between the expression of 100s of miRNAs across comparative samples. This technique also allows the quantification of the expression of targeted miRNAs to validate observations determined by whole-genome screening or to analyze few specific miRNAs on a large number of samples. Here, we describe the validation of a method published by ABIs on their web site allowing to reverse transcribe and pre-amplify multiple miRNAs and snoRNAs simultaneously. The validation of this protocol was performed on human muscle and plasma samples. Fast and cost efficient, this method achieves an easy and convenient way to screen a relatively large number of miRNAs in parallel.

The effect of known cardiovascular risk factors on carotid-femoral pulse wave velocity in school-aged children: a population based twin study

Childhood cardiovascular risk factors affect vascular function long before overt cardiovascular disease. Twin studies provide a unique opportunity to examine the influence of shared genetic and environmental influences on childhood cardiovascular function. We examined the relationship between birth parameters, markers of adiposity, insulin resistance, lipid profile and blood pressure and carotid-femoral pulse wave velocity (PWV), a validated non-invasive measure of arterial stiffness in a healthy cohort of school-aged twin children. PWV was performed on a population-based birth cohort of 147 twin pairs aged 7-11 years. Fasting blood samples, blood pressure and adiposity measures were collected concurrently. Mixed linear regression models were used to account for twin clustering, within- and between-twin pair associations. There were positive associations between both markers of higher adiposity, insulin resistance, elevated triglycerides and PWV, which remained significant after accounting for twin birth-set clustering. There was a positive association between both diastolic and mean arterial blood pressure and PWV in within-pair analysis in dizygotic, but not monozygotic twins, indicating genetic differences evident in dizygotic not monozygotic twins may affect these associations. Increased blood pressure, triglycerides and other metabolic markers are associated with increased PWV in school-aged twins. These results support both the genetic and environmental contribution to higher PWV, as a marker of arterial stiffness, and reiterate the importance of preventing metabolic syndrome from childhood.

Blood pressure and cognitive function: the role of central aortic and brachial pressures

Central (aortic) blood pressures differ from brachial pressures and may be more relevant to the study of cognitive function, given that blood is delivered to the brain through the central large arteries. Pulse-pressure amplification reflects the augmentation of blood pressure between the central and peripheral arteries, which diminishes with aging. We aimed to determine the association between central blood pressure and cognitive function in independently living adults aged 20 to 82 years (N = 493). In adjusted regression models, higher central systolic pressure and higher central pulse pressure were each associated with poorer processing speed, Stroop processing, and recognition memory. Lower amplification was associated with poorer Stroop processing, working memory, and recognition memory. Higher brachial systolic pressure and brachial pulse pressure were both associated with poorer Stroop processing. In summary, central pressures and amplification were sensitive indicators of cognitive aging, predicting aspects of cognitive performance not predicted by brachial blood pressure.