Standardized, automated procedure for measurement of serum hemolytic complement activity

[This corrects the article on p. 1635 in vol. 16.].     

Development of an automated procedure for fluorescent DNA sequencing

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.     

Ecology,song similarity and phylogeny predict natural hybridization in an avian family

Hybridization is important in the evolution of many animal groups; however, broad scale patterns of natural hybridization are still poorly understood. Using phylogenetic comparative analyses, we tested for relationships between demographic, ecological, phenotypic and phylogenetic variables and the incidence of natural hybridization among 45 species of North American wood warbler. Since 1980, hybrids have been documented in 24 species (53 %). We detected negative relationships between the incidence of hybridization and both breeding range size and phylogenetic distance, and positive relationships between the incidence of hybridization and (1) song similarity, (2) the extent of breeding sympatry of species pairs and (3) the number of additional heterospecific warbler species that co-occur during breeding. Neither population size nor breeding habitat quality (as measured by threats to survival or reproduction in breeding areas) explained variation in the incidence of hybridization. Our results suggest a potential role of limited breeding habitat in wood warbler hybridization events and a net positive effect of breeding sympatry. They also support the hypothesis that mating signal similarity facilitates hybridization events and are consistent with an increase in reproductive isolation with increasing genetic divergence. Our results also suggest the presence of phylogenetic signal in wood warbler hybridization. By investigating natural hybridization at the taxonomic level of family, we have identified several previously undocumented patterns of natural hybridization. This study demonstrates the utility of examining the combined effects of ecology, demography, phenotype and phylogeny when studying variation in the expression of natural hybridization among taxa.     

Navigating molecular space for reaction mechanisms: an efficient,automated procedure

Mechanism is a core chemical concept that has vital implications for reaction rate, efficiency and selectivity. The discovery of mechanism is not easy due to the great diversity of possible chemical rearrangements in even relatively simple systems. For this reason, mechanisms involving bond breaking and forming are usually proposed via chemical intuition – which limits the scope of considered possibilities – and these hypotheses are then tested using simulation or experiment. This article discusses an automated simulation strategy for investigating multiple elementary step reaction mechanisms in chemical systems. The method starts from a single input structure and seeks out nearby intermediates, optimises the proposed structures and then determines the kinetic viability of each elementary step. The kinetically accessible intermediates are catalogued and new searches are performed on each unique structure. This process is repeated for an arbitrary number of steps without human intervention, and massively parallel computation enables fast searches in chemical space. Importantly, this strategy can be empirically shown to lead to a finite number of accessible structures, not a combinatorial explosion of intermediates. Therefore, the method should be able to predict multi-step reaction pathways in many interesting chemical systems. Demonstrations on organic reactions and a hydrogen storage material, ammonia borane, show that the herein proposed strategy can uncover complex reactivity without relying on existing chemical intuition.     

Crystallization of blood coagulation factor XIII by an automated procedure

Both recombinant blood coagulation factor XIII alpha-chain and factor XIII isolated from human placenta have been crystallized using a novel robotic system for the automatic screening of crystallization conditions. The monoclinic and orthorhombic crystals obtained are suitable for X-ray analysis.     

An automated procedure for the determination of oxytetracycline

An automated analytical system has been used to determine the amount of oxytetracycline in pharmaceutical products and fermentation broth at a level of 0.1mg/mL. The method is based on the formation of a colour complex between the oxytetracycline and ammonium molybdate in acid media at 80°C. The effect of temperature, concentration of HCl and ammonium molybdate upon the stability of the complex was determined. Standard error of difference is 0.43% for pharmaceutical products and the fermentation broth.     

Plasma lipid hydroperoxides measurement by an automated xylenol orange method

Lipid hydroperoxides (LH) appear to be good candidates as initial biomarkers of oxidative stress. We describe an automated method to quantify it, based on a known principle: oxidation of Fe II to Fe III by lipid hydroperoxides, under acidic conditions, followed by complexation of Fe III by xylenol orange. This method requires only a 10-microl sample volume of heparinized plasma or serum. It has been carried out automatically, with two reagents, in a two-end-point mode with bichromatic detection at 570 and 700 nm. The within-run precision, measured on a low- and a high-level plasma, was 5.0+/-0.3 and 14.0+/-0.6 microM (n=25 for each series). The between-run precision (one run for 18 days), evaluated on two commercial controls, was 5.6+/-0.5 microM (CV=8.9%) and 7.9+/-0.5 microM (CV=6.3%). The recovery of known amounts of tert-butylhydroperoxide (1 and 2 microM) added to human plasma was 98%. The specificity was demonstrated by the excellent correlation of the values of 42 samples measured either directly, with a simple dilution, or after gel permeation chromatography. The reference interval determined on 21 subjects was 4.9+/-1.7 microM. This was in the upper range of previously published values but our recovery and chromatographic experiments strongly suggest that former methods have underestimated the true content of LH in human plasma.     

Evaluation of an automated fluorescent antibody procedure for detection of Salmonella in foods and feeds.

A prototype automated system using fluorescent antibody (FA) was evaluated for rapid detection of salmonellae in foods. Samples were enriched in selenite cystine and tetrathionate broths. After incubation, both were transferred into fresh selenite cystine for a 4-h "post-enrichment" to dilute possible background fluorescence from product. These cultures were then analyzed automatically, and results were compared with those obtained by the methods of the Association of Official Analytical Chemists (AOAC). Initially, 167 samples of milk powder, dried yeast, and imported frog legs were examined. The AOAC and automated FA methods correlated well with all samples but frog legs. Difficulty with the latter was caused by procedural and mechanical problems coupled with high numbers of competing microorganisms in post-enrichment cultures. Modification of procedure and partial redesign of equipment corrected these difficulties, and excellent correlation was obtained with another 116 frog leg samples. All 89 AOAC-confirmed positives were also detected by the automated FA method, and there were only 4% false FA positives. The system shows potential for screening products for salmonellae; however, all positives should be confirmed by manual biochemical and serological methods.     

A quantitative histochemical procedure for measurement of starch in apple fruits

Measurements of starch (e.g. amyloplasts in stomatal guard cells, sieve elements, root tips or the starch sheath) is often very difficult using most analytical methods. An evaluation was made of interactive computer image analysis of starch measurements in apple fruits. The results obtained indicate that quantitative histochemistry can be an appropriate method to quantify starch. Correlations for starch values between the image analysis system and a colorimetric system were quantified. The thickness of plastic-embedded slices had no influence on the accuracy of the area occupied by image-quantified starch (starch/slice) or on its variance. The magnification of the objective also had no effect on measured starch-occupied areas (starch/slice), but there were big differences in variance. The number of replications required to establish statistically significant differences were calculated.     

Quantitative determination of aliphatic diamines and polyamines by an automated liquid chromatography procedure

The naturally occurring aliphatic polyamines and diamines can be separated with an improved automated procedure on a sulfonated-type ion-exchange column. Quantitative determinations can be carried out on 2–20 nmoles of each amine.In addition to the simple diamines, spermidine, and spermine, this procedure separates a number of naturally occurring derivatives, such as monoacetyl-1,4-diaminobutane, monoacetylspermidine, carbamyl-1,4-diaminobutane, 1,4-diamino-2-hydroxybutane, and glutathionylspermidine.The method can be used for a variety of biological materials, including urine, animal tissues, and bacterial extracts.     

A marker-based measurement procedure for unconstrained wrist and elbow motions.

A protocol is proposed to obtain the joint angles of wrist and elbow from tracked triads of surface markers on each limb segment. Cuffs placed on the limb support the rigidity of the triads. Additional markers are used to mark the approximate positions of joints. Corrections of surface marker data for skin motion are derived from a priori knowledge about plausible joint motions. In addition, ill-conditioned states are trapped when the elbow is nearly fully extended. The protocol is applied to sample motions which demonstrate the use and the effect of the corrections. The results show that the model assumptions are reasonable and that accurate joint rotations can be obtained. The correction steps prove to be an essential part of upper-extremity movement analysis.     

MODIP revisited: re-evaluation and refinement of an automated procedure for modeling of disulfide bonds in proteins

There have been several attempts to stabilize proteins through the introduction of engineered disulfide bonds. For reasons that are currently unclear, these have met with mixed success. Hence identification of locations where introduction of a disulfide cross-link will lead to protein stabilization is still a challenging task. A computational procedure, MODIP, was introduced more than a decade ago to select sites in protein structures that have the correct geometry for disulfide formation when replaced by Cys. In this study, we re-evaluated the stereochemical criteria used by MODIP for the selection and gradation of sites for modeling disulfides. We introduced steric criteria to check for energetically unfavorable non-bonded contacts with the modeled disulfide, since these can considerably offset the stabilizing effect of the cross-link. The performance of the refined procedure was checked for its ability to correctly predict naturally occurring disulfide bonds in proteins. A set of proteins in which disulfide bonds were introduced experimentally were analyzed with respect to MODIP predictions, stability and other parameters such as accessibility, residue depth, B-factors of the mutated sites, change in volume upon mutation and loop length enclosed by the disulfide. The analysis suggests that in addition to proper stereochemistry, stabilizing disulfides occur in regions of low depth, relatively high mobility, have a loop length greater than 25 and where the disulfide typically occupies a volume less than or equal to that of the original residues.