Stokes's cradle: Newton's cradle with liquid coating.

Granular flows involving liquid-coated solids are ubiquitous in nature (pollen capture, avalanches) and industry (filtration, pharmaceutical mixing). In this Letter, three-body collisions between liquid-coated spheres are investigated experimentally using a "Stokes's cradle," which resembles the popular desktop toy Newton's cradle (NC). Surprisingly, previous work shows that every possible outcome was observed in the Stokes's cradle except the traditional NC outcome. Here, we experimentally achieve NC via guidance from a theory, which revealed that controlling the liquid-bridge volume connecting two target particles is the key in attaining the NC outcome. These three-body experiments also provide direct evidence that the fluid resistance upon rebound cannot be completely neglected due to presumed cavitation; this resistance also influences two-body systems yet cannot be isolated experimentally in such systems.

Long-term response to subtotal colectomy in colonic inertia.

The purpose of this study was to determine the long-term outcome of patients who had previously undergone subtotal colectomy for severe idiopathic constipation at the University of Florida between 1983 and 1987. In addition, we aimed to determine whether preoperative motility abnormalities of the upper gastrointestinal tract are more common among those patients who have significant postoperative complications after subtotal colectomy. We evaluated 13 patients who underwent subtotal colectomy for refractory constipation between 1983 and 1987 at the University of Florida. Preoperatively, all patients exhibited a pattern consistent with colonic inertia as demonstrated by means of radiopaque markers. Each patient was asked to quantitate the pain intensity and frequency of their bowel movements before and after surgery. In seven patients an ileosigmoid anastomosis was performed, whereas in six patients an ileorectal anastomosis was used. Abdominal pain decreased after subtotal colectomy. Patients with abnormal upper gastrointestinal motility preoperatively experienced greater postoperative pain than those with normal motility regardless of the type of anastomosis. In addition, the number of postoperative surgeries was similar in those patients with abnormal upper motility compared to those with normal motility. Overall, the total number of bowel movements per week increased from 0.5 +/- 0.03 preoperatively to 15 +/- 4.5 (P < 0.007) postoperatively. The results of our study suggest that patients with isolated colonic inertia have a better long-term outcome from subtotal colectomy than patients with additional upper gastrointestinal motility abnormalities associated with their colonic inertia.

Droplet growth by coalescence in binary fluid mixtures.

The evolution of the drop-size distribution in immiscible fluid mixtures following well-specified shear histories is investigated by in situ microscopy, allowing determination of the shear-induced coalescence efficiency epsilon. At small capillary number Ca, epsilon is constant, whereas at larger values of Ca, epsilon decreases, in agreement with theory accounting for slight deformation of the drops in close approach. Coalescence causes the drop-size distribution to broaden in general, but greater deformation of the larger drops at high shear rates causes the drop-size distribution to remain narrow.

Cellulase recovery via membrane filtration.

A combined sedimentation and membrane filtration process was investigated for recycling cellulase enzymes in the biomass-to-ethanol process. In the first stage, lignocellulose particles longer than approx 50 microm were removed by means of sedimentation in an inclined settler. Microfiltration was then utilized to remove the remaining suspended solids. Finally, the soluble cellulase enzymes were recovered by ultrafiltration. The permeate fluxes obtained in microfiltration and ultrafiltration were approx 400 and 80 L/(m2 x h), respectively. A preliminary economic analysis shows that the cost benefit of enzyme recycling may be as much as 18 cents/gal of ethanol produced, provided that 75% of the enzyme is recycled in active form.

Optimization of repeated-batch transcription for RNA production.

A major obstacle to large-scale RNA production is the high raw material cost. This work focuses on reducing the cost of RNA produced by in vitro transcription. RNA can be produced by transcription from DNA templates immobilized on solid supports such as agarose beads, with yields comparable to traditional solution-phase transcription. The advantage of immobilized DNA is that the templates can be recovered from the reaction and reused in multiple rounds, eliminating unnecessary disposal. Additionally, approximately 50% of the original RNA polymerase added to the reaction is also recovered in active form with the DNA and can be used for further rounds of repeated-batch transcription. Thus, adding only a fraction of the first-round enzyme concentration to subsequent rounds is sufficient for maintaining yields comparable to batch reactions for many rounds, with lowered cost. Results for two different DNA templates support a simplified model for repeated-batch transcription, based on the previous work of Davis and Breckenridge (J Biotechnol 1999;71:25-37). The model successfully predicts the yields for several of rounds of repeated-batch transcription using various enzyme addition schemes, and it was used to optimize the process by reducing the cost of raw materials per amount of RNA produced by 40-70%.

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product.

S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t0.5 approximately 10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K0.5 approximately 100 microM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine.

Polyamine regulation of ornithine decarboxylase synthesis in Neurospora crassa.

Ornithine decarboxylase (ODC) of the fungus Neurospora crassa, encoded by the spe-1 gene, catalyzes an initial and rate-limiting step in polyamine biosynthesis and is highly regulated by polyamines. In N. crassa, polyamines repress the synthesis and increase the degradation of ODC protein. Changes in the rate of ODC synthesis correlate with similar changes in the abundance of spe-1 mRNA. We identify two sequence elements, one in each of the 5' and 3' regions of the spe-1 gene of N. crassa, required for this polyamine-mediated regulation. A 5' polyamine-responsive region (5' PRR) comprises DNA sequences both in the upstream untranscribed region and in the long 5' untranslated region (5'-UTR) of the gene. The 5' PRR is sufficient to confer polyamine regulation to a downstream, heterologous coding region. Use of the beta-tubulin promoter to drive the expression of various portions of the spe-1 transcribed region revealed a 3' polyamine-responsive region (3' PRR) downstream of the coding region. Neither changes in cellular polyamine status nor deletion of sequences in the 5'-UTR alters the half-life of spe-1 mRNA. Sequences in the spe-1 5'-UTR also impede the translation of a heterologous coding region, and polyamine starvation partially relieves this impediment. The results show that N. crassa uses a unique combination of polyamine-mediated transcriptional and translational control mechanisms to regulate ODC synthesis.

Modeling of repeated-batch transcription for production of RNA.

Enzymatic transcription for in vitro production of ribonucleic acids (RNA) is typically carried out in small batch reactors which suffer from low yields and high costs due to the formation of abortive RNA transcripts and the disposal of the DNA template and polymerase enzyme after a single use. This work considers repeated-batch transcription in which the DNA template molecules are immobilized on beads which are recovered and reused in multiple rounds. Some of the enzyme binds to the DNA template and is also recovered and reused. A model of this process is presented which employs equilibrium binding between the enzyme and template and which includes a first-order sequential deactivation of the enzyme. The model predicts the yields of RNA product and aborts for each round of repeated-batch transcription with no DNA addition after the first round and only partial enzyme replacement. The yield of RNA product per substrate (nucleoside triphosphates) generally decreases in subsequent rounds, whereas the yields of RNA product per enzyme and per template increase due to their reuse. Experimental data are presented which confirm the model and which show how the model parameters are obtained. A cost analysis shows that the cost of RNA production can be reduced by more than 50% for the system tested by employing an optimum number of rounds of repeated-batch transcription.

An epidemiological evaluation of the use of microbiological tools for identifying gonorrhoea infection networks.

We aimed to assess the utility of various techniques for identifying gonorrhoea infection networks. All residents of a non-metropolitan North Carolina county visiting a sexually transmitted disease (STD) clinic during a 17-month period were screened for gonorrhoea. Infection networks were estimated by serovar type combined with antibiotic resistance, arbitrarily primed polymerase chain reaction (AP-PCR), or temporal clustering. The residential addresses of infected patients were geocoded and mapped. Among 2 serovar types, the presence of distinguishing characteristics of a network, based on questionnaire data, was assessed with prevalence ratios and 95% confidence intervals (CIs) relative to those not in the network. Twenty-five serovar types were identified among 759 gonorrhoea infections. In one serovar, the networks further delineated by temporal clusters correlated with particular AP-PCR types. In most instances, however, different typing techniques painted different network pictures. No refined serovar network stood out as having a particular set of characteristics that could be used to shape intervention. Teasing out an individual infection network with unique characteristics will require the development and use of other microbiological tools.

Application of a fed-batch system to produce RNA by in vitro transcription.

A novel fed-batch method is presented for the production of RNA by in vitro transcription performed in a stirred-cell reactor with pH-controlled addition of reaction components. Solution equilibrium analysis is applied to determine the ratios of feed components (including nucleoside triphosphates (NTPs), magnesium salt, and base) which allow the desired NTP concentrations, free magnesium concentration, and pH to be maintained during the reaction. Results are presented for fed-batch and batch reactions performed with two DNA templates encoding a 12mer RNA and a 38mer RNA. For the dodecamer RNA, the fed-batch mode is only modestly better than batch reactions, with no significant increase in the efficiency of NTP incorporation but with 40% improvement in the amount of RNA produced per unit of polymerase or DNA. For the 38mer, fed-batch transcription provides a substantial increase in the efficiency of NTP incorporation and 100% improvement in the production of RNA per unit of polymerase or DNA. Cost analyses are presented which show how optimal NTP concentrations in batch and fed-batch reactions will be dependent on the relative costs of NTPs, T7 RNA polymerase, and DNA templates for the particular application. The use of a fed-batch mode appears to have the potential for substantial improvement in the economics of RNA production for at least some RNA sequences.

Longitudinal evaluation of serovar-specific immunity to Neisseria gonorrhoeae.

The serovars of Neisseria gonorrhoeae that are predominant in a community change over time, a phenomenon that may be due to the development of immunity to repeat infection with the same serovar. This study evaluated the epidemiologic evidence for serovar-specific immunity to N. gonorrhoeae. During a 17-month period in 1992-1994, all clients of a sexually transmitted disease clinic in rural North Carolina underwent genital culture for N. gonorrhoeae. Gonococcal isolates were serotyped according to standard methods. Odds ratios for repeat infection with the same serovar versus any different serovar were calculated on the basis of the distribution of serovars in the community at the time of reinfection. Of 2,838 patients, 608 (21.4%; 427 males and 181 females) were found to be infected with N. gonorrhoeae at the initial visit. Ninety patients (14.8% of the 608) had a total of 112 repeat gonococcal infections. Repeat infection with the same serovar occurred slightly more often than would be expected based on the serovars prevalent in the community at the time of reinfection, though the result was marginally nonsignificant (odds ratio = 1.5, 95% confidence interval 1.0-2.4; p = 0.05). Choosing partners within a sexual network may increase the likelihood of repeat exposure to the same serovar of N. gonorrhoeae. Gonococcal infection did not induce evident immunity to reinfection with the same serovar.