Efficient random subcloning of DNA sheared in a recirculating point-sink flow system

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.     

The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus

An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.     

An Experimental Investigation into the Feed Rate Capacity of Rotary Valve and Blow Tank Feeders

Industry widely uses rotary valves and blow tanks for the pneumatic conveying of products, each having their pros and cons depending on the specific application. This article shows the differing results obtained when low-velocity conveying a product through a common pipeline using both a drop-through rotary valve and a bottom-discharge blow tank feeder. A number of issues arise in the rotary valve system, the main one being rotary valve air leakage. A blow tank system, on the other hand, does not leak, as it is an enclosed system. The experimental results show dramatic differences in product throughput. Further exploration leads to a novel modification being made to the rotary valve system in an attempt to increase its capacity. The result of this modification shows a slight increase in output tonnage, but still significantly less than that obtained from the blow tank.     

P#: a concurrent Prolog for the .NET framework

We discuss P#, our implementation of a tool that allows interoperation between a concurrent superset of the Prolog programming language and C#. This enables Prolog to be used as a native implementation language for Microsoft's .NET platform. P# compiles a linear logic extension of Prolog to C# source code. We can thus create C# objects from Prolog and use C#'s graphical, networking and other libraries. We add language constructs on the Prolog side that allow concurrent Prolog code to be written. A primitive predicate is provided that evaluates a Prolog structure on a newly forked thread. Communication between threads is based on the unification of variables contained in such a structure. It is also possible for threads to communicate through a globally accessible table. All of the new features are available to the programmer through new built-in Prolog predicates. We discuss two software engineering tools implemented using P#. Copyright © 2004 John Wiley & Sons, Ltd.     

Collaborative cross-checking to enhance resilience

Resilience, the ability to adapt or absorb disturbance, disruption, and change, may be increased by team processes in a complex, socio-technical system. In particular, collaborative cross-checking is a strategy where at least two individuals or groups with different perspectives examine the others’ assumptions and/or actions to assess validity or accuracy. With this strategy, erroneous assessments or actions can be detected quickly enough to mitigate or eliminate negative consequences. In this paper, we seek to add to the understanding of the elements that are needed in effective cross-checking and the limitations of the strategy. We define collaborative cross-checking, describe in detail three healthcare incidents where collaborative cross-checks played a key role, and discuss the implications of emerging patterns.     

Pigeon perception and discrimination of rapidly changing texture stimuli

The perception and discrimination of rapidly changing texture stimuli by pigeons was examined in a target localization task. Five experienced pigeons were rewarded for finding and pecking at a randomly placed odd target block of small repeated elements embedded in a larger rectangular array of contrasting distractor elements. On dynamic color test trials, the color of the target, distractor, or both of these regions changed at rates of 100, 250, 500, or 1000 ms per frame. The number of colors appearing within such trials also varied. Pigeons performed well above chance in all test conditions, with target-associated changes producing the best discrimination. The results suggest: (a) global relational information can exclusively guide target localization behavior, (b) pigeons can perceptually group and segregate colored textured differences quite rapidly (< or = 100 ms), and (c) pigeons may possess automatic search control processes that can be captured by stimulus-driven changes in the display.     

Effects of cocaine on dopamine receptor gene expression: a study in the postmortem human brain

The effects of chronic cocaine exposure on dopamine D1 and D2 receptor gene expression in the human brain were studied in postmortem samples from chronic cocaine abusing and matched control subjects. Using in situ hybridization of receptor autoradiography to examine messenger ribonucleic acid (RNA) and binding sites, respectively, neither D1 nor D2 receptor expression was found to be changed in the nucleus accumbens, caudate, putamen, or substantia nigra of the cocaine-exposed subjects. Although chronic cocaine exposure can produce alterations in dopaminergic neurotransmission, sustained compensatory changes in dopamine receptor expression do not appear to occur in the human.