A Systematic Structure–Activity Study of a New Type of Small Peptidic Transfection Vector Reveals the Importance of a Special Oxo-Anion-Binding Motif for Gene Delivery

We discovered a new class of artificial peptidic transfection vectors based on an artificial anion-binding motif, the guanidiniocarbonylpyrrole (GCP) cation. This new type of vector is surprisingly smaller than traditional systems, and our previous work suggested that the GCP group was important for promoting critical endosomal escape. We now present here a systematic comparison of similar DNA ligands featuring our GCP oxo-anion-binding motif with DNA ligands only consisting of naturally occurring amino acids. Structure–activity studies showed that the artificial binding motif clearly outperformed natural amino acids such as histidine, lysine, and arginine. It improved the ability to shuttle foreign genetic material into cells, yet successfully mediated endosomal escape. Also, plasmids that were complexed by our artificial ligands were stabilized against cytosolic degradation to some extent. This resulted in the successful expression of plasmid information (comparable to gold standards such as polyethyleneimine). Hence, our study clearly demonstrates the importance of the tailor-made GCP anion-binding site for efficient gene transfection.     

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Fractal fragmentation of rocks within sturzstroms: insight derived from physical experiments within the ETH geotechnical drum centrifuge

An investigation of the behaviour and energy budget of sturzstroms has been carried out using physical, analytical and numerical modelling techniques. Sturzstroms are rock slides of very large volume and extreme run out, which display intensive fragmentation of blocks of rock due to inter-particle collisions within a collisional flow. Results from centrifugal model experiments provide strong arguments to allow the micro-mechanics and energy budget of sturzstroms to be described quantitatively by a fractal comminution model. A numerical experiment using a distinct element method (DEM) indicates rock mass and boundary conditions, which allow an alternating fragmenting and dilating dispersive regime to evolve and to sustain for long enough to replicate the spreading and run out of sturzstroms without needing to resort to peculiar mechanism. The fragmenting spreading model supported here is able to explain the run out of a fluid-absent granular flow beyond the travel distance predicted by a Coulomb frictional sliding model. This, and its strong relation to internal fragmentation, suggests that a sturzstrom constitutes a landslide category of its own. This study provides a novel framework for the understanding the physics of such sturzstroms.     

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co‐expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5′‐gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast‐enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast‐enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon‐optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest. Copyright © 2009 John Wiley & Sons, Ltd.     

The Design and Evaluation of a Computerized and Physical Simulator for Training Clinical Prostate Exams

The most effective screening for prostate cancer combines the prostate specific antigen blood test with the digital rectal examination (DRE). In performing a DRE, two sequential tasks are completed: ( task a) palpating the prostate to identify abnormalities and ( task b) linking identified abnormalities to a disease diagnosis. At present, clinicians find too few abnormalities and have variable rates of detection, due in part to the inadequacy of training simulators. The Virginia Prostate Examination Simulator (VPES) was designed, built, and tested to address the inadequacies of current simulators by incorporating the design requirements of the basic elements of accurate anatomy, multiple and reconfigurable scenarios of graded difficulty, and technique and performance feedback. We compared the training effectiveness of the VPES with two commercial simulators in an experiment of 36 medical and nurse practitioner students. Results indicate each type of training simulator-improved abilities, in general. Upon closer analysis, however, the following key patterns emerge: 1) Across all types of training, more deficiencies lie in skill-based rather than rule-based decision making, which improves only for VPES trainees; 2) only VPES training transfers both to other simulators and previously unencountered scenarios; 3) visual feedback may increase the number of abnormalities reported yet hinder the ability to discriminate; and 4) applied finger pressure did not correlate with the ability to identify abnormalities.      

Regional cerebral volume loss associated with verbal learning and memory in dementia of the Alzheimer type.

Twenty-seven research participants with dementia of the Alzheimer type were studied with the California Verbal Learning Test (D. C. Delis, J. H. Kramer, E. Kaplan, & B. A. Ober, 1987) and standardized volume measures of the mesial temporal cortical gray matter, neocortical gray matter, thalamus, and caudate nuclei, from magnetic resonance imaging. A pattern of atrophic brain changes in the mesial temporal lobes (MTL) and the thalamus, with relatively less severe atrophy in the neocortical gray matter, was associated with poorer learning of the word list. Similar patterns of brain atrophy were observed for measures of delayed recall and recognition hits. However, for delayed recall, neither contribution was statistically significant, and for recognition hits, MTL was only at the trend level for significance. These results provide evidence that the verbal memory deficit of Alzheimer's disease (AD) is associated not only with the mesial temporal limbic cortex, thought to be the site of earliest and most severe pathology in AD, but also with damage in the thalamus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Optimizing Reaction Conditions of the NanoOrange® Protein Quantitation Method for Use With Microplate-based Automation

Because of the rapidly expanding need for higher sample throughput in drug discovery, automation of corresponding biochemical analyses is desirable. In particular, automation of protein quantitation is crucial since its results are used extensively. Recently, a single-reagent fluorescent protein quantitation method (NanoOrange®) with attractive performance attributes has become available. While it can potentially be automated with liquid handling workstations, several of this method's reaction parameters need to be optimized.

We studied the time and a temperature dependence of the NanoOrange protein quantitation reaction in ninety-six well black microplates using either a temperature-regulated hot block or a microwave oven as heat sources. Fluorescence of the NanoOrange reaction was quantified with a multimode microplate spectral scanner.

Time-dependent heating profiles of filled microplates placed on hot blocks at fixed temperatures (45, 55, 65, 75, and 95°C) revealed temperature differences of 4–7°C cooler for the outside wells compared to the inner wells, however the maximum well temperature did not exceed 65°C. Similar time-temperature studies of microwave-heated microplates revealed an equilibrium temperature of 45–49°C that was 10–16°C lower than microplates that were block heated.

The bovine serum albumin (BSA): NanoOrange standard curves created using a hot block increased in slope from 45°C to 55°C, but then remained constant from 65 to 85°C.

Fluorescence of the BSA: NanoOrange standard curve created using a microwave oven was about half the magnitude of the hot block-derived curves, possible reflecting a lower energy transfer rate of the microwave oven. We conclude that the NanoOrange protein quantitation method can be automated if a microplatecompatible hot block with a 65-85°C surface can heat the microplate for minimum of 15 min prior to quantifying the reaction's fluorescence.     

Coping through emotional approach: Scale construction and validation.

Four studies demonstrate the psychometric adequacy and validity of scales designed to assess coping through emotional approach. In separate undergraduate samples, exploratory and confirmatory factor analyses of dispositional (Study 1) and situational (Study 3) coping item sets yielded 2 distinct emotional approach coping factors: emotional processing (i.e., active attempts to acknowledge and understand emotions) and emotional expression. The 2 scales yielded high internal consistency and test-retest reliability, as well as convergent and discriminant validity. A study (Study 2) of young adults and their parents established the scales' interjudge reliabilities. Longitudinal (Study 3) and experimental (Study 4) research supported the predictive validity of the emotional approach coping scales with regard to adjustment to stressful encounters. Findings highlight the utility of functionalist theories of emotion as applied to coping theory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)