Removal of TSE agent from plasma products manufactured in the United Kingdom.

BACKGROUND AND OBJECTIVES: The outbreak of vCJD in the UK leads to concern regarding the potential for human-to-human transmission of this agent. Plasma-derived products such as albumin, immunoglobulin and coagulation factors were manufactured by BPL from UK plasma up until 1999 when a switch to US plasma was made. In the current study, the capacity of various manufacturing processes that were in use both prior to and after this time to remove the TSE agent was tested. MATERIALS AND METHODS: Small-scale models of the various product manufacturing steps were developed. Intermediates were spiked with scrapie brain extract and then further processed. Samples were assayed for the abnormal form of prion protein (PrP(SC) ) by Western blotting, and the reduction in the amount of scrapie agent determined. RESULTS: Many of the manufacturing process steps produced significant reduction in the scrapie agent. Particularly effective were steps such as ethanol fractionation, depth filtration, ion-exchange and copper chelate affinity chromatography. Virus retentive filters, of nominal pore size 15 or 20 nm, removed >3 log. The total cumulative reduction capacity for individual products was estimated to range from 7 to 14 log. In the case of factor VIII (8Y), the total removal was limited to 3 log. CONCLUSION: All the processes showed a substantial capacity to remove the TSE agent. However, this was more limited for the intermediate purity factor VIII 8Y which included fewer manufacturing steps.

Adaptive evolution in subterranean termite antifungal peptides.

We identified and analysed mRNA sequences of two immune proteins from the subterranean termites Reticulitermes flavipes and Reticulitermes virginicus. These proteins correspond to two immune proteins described in the distantly related termite genus Nasutitermes; termicin, which is a small antifungal peptide, and GNBP2, which functions both as a broad pattern recognition receptor and a direct antifungal effector. A population genetic analysis of nucleotide intraspecific polymorphism and interspecific divergence indicates that a selective sweep has reduced polymorphism in the termicins. Moreover, this selective sweep appears to have been driven by the positive selection of beneficial amino acid changes in the antifungal peptide. In contrast, the pattern of polymorphism and divergence in GNBP2 corresponds to the standard neutral model of evolution.

Impact of clarification strategy on chromatographic separations: Pre-processing of cell homogenates.

This research focused on how the extent and type of primary solid-liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original "soluble" state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration.

Performance prediction of industrial centrifuges using scale-down models.

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.

Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions.

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.

The evolution of anisogamy: a game-theoretic approach.

A popular theory has proposed that anisogamy originated through disruptive selection acting on an ancestral isogamous population, though recent work has emphasized the importance of other factors in its evolution. We re-examine the disruptive selection theory, starting from an isogamous population with two mating types and taking into account the functional relationship, g(m), between the fitness of a gamete and its size, m, as well as the relationship, f(S), between the fitness of a zygote and its size, S. Evolutionary game theory is used to determine the existence and continuous stability of isogamous and anisogamous strategies for the two mating types under various models for the two functions g(m) and f(S). In the ancestral unicellular state, these two functions are likely to have been similar; this leads to isogamy whether they are sigmoidal or concave, though in the latter case allowance must be made for a minimal gamete size. The development of multicellularity may leave g(m) relatively unchanged while f(S) moves to the right, leading to the evolution of anisogamy. Thus, the disruptive selection theory provides a powerful explanation of the origin of anisogamy, though other selective forces may have been involved in the subsequent specialization of micro- and macrogametes.

Hydrophobic interaction ligand selection and scale-up of an expanded bed separation of an intracellular enzyme from Saccharomyces cerevisiae.

A prototype Streamline-Phenyl matrix was evaluated in a hydrophobic interaction mode for the direct recovery of alcohol dehydrogenase (ADH) from yeast cell homogenate. At 5% breakthrough of ADH, a yield of 100% was obtained for a dynamic expanded bed capacity of 240 U(ADH)/ml matrix with a purification factor of 9.2. This compared with a dynamic capacity of 3013 U(ADH)/ml matrix for the packed bed equivalent and a purification factor of 18. In both systems the purification factor was found to increase simultaneously with a decrease in yield as the load of homogenate or breakthrough of ADH was increased. The expanded bed mode of operation conferred considerable robustness with respect to process fouling. No loss in yield was seen over five cycles of repeat loading with an unclarified homogenate. By contrast the packed bed media showed a decrease in yield from 86 to 56% over the same period. Successful scale up of the expanded bed protocol for a 20% breakthrough was demonstrated over a fourfold increase in column diameter. The application of hydrophobic interaction chromatography mediated expanded bed adsorption and its scale-up is discussed in the context of large-scale operations.

Improving the view in the rectal clinic: a randomised control trial.

BACKGROUND: Rigid sigmoidoscopy forms an integral part of the out-patient assessment of patients with colorectal symptoms. However, the value of this of this examination is often diminished by faecal loading of the rectum. This trial aimed to determine the ability of a single self-administered glycerine suppository to clear the rectum in preparation for rigid sigmoidoscopy, and considered patient acceptability of this practice. METHODS: Consecutive patients were randomly allocated to receive suppository or no suppository prior to out-patient rigid sigmoidoscopy. Assessment was made of patient compliance, the effectiveness of rectal examination, and the depth to which the sigmoidoscope was inserted. RESULTS: 131 patients were randomised into suppository (n = 66) or control groups (n = 65). The number of patients deemed to have good views of the rectum (> 75% of rectal mucosa seen) was significantly greater in suppository than control groups (79% versus 26.2%, P < 0.05 Chi square test), whilst that of poor examinations (< 50% of rectal mucosa seen) was significantly greater in control than suppository groups (44.6% versus 4%, P < 0.05). The depth of insertion of the sigmoidoscope was significantly greater in those receiving suppositories (54.5% versus 21.5% undergoing evaluation to 18 cm or more, P < 0.05). Compliance amongst those who received suppositories was high with only 3 of 53 (4.5%) patients in the suppository group evaluated by questionnaire reporting difficulty or concerns over their use. CONCLUSION: Self-administered suppositories are acceptable to patients and significantly improve the efficiency of outpatient rigid sigmoidoscopy. Their usage should become routine.

Laboratory scaledown of protein purification processes involving fractional precipitation and centrifugal recovery.

The ability to predict the performance of large-scale processes is central to the rapid development of successful operations at the pilot and industrial scale. In this article, we examine the operation, at laboratory scale, of precipitation reactors and centrifuges for protein precipitate recovery and dewatering and how they might best mimic large-scale reactors and centrifuges, in this case, a pilot-scale batch stirred-tank reactor and a multichamber-bowl centrifuge. Novel approaches to bench-top centrifuge operation are provided, in particular with a view to delivery of material for subsequent high-resolution purification, which would be obtained at full pilot scale. Results are presented in terms of properties of the protein precipitates, the fraction of solids recovered, and the extent of dewatering achieved. Good agreement was obtained at bench scale (a 1000-fold scale down factor) for all of these parameters for pilot-scale, batch-feed operation. In addition, the methodology developed allows identification of the extent of break-up that occurs in continuous-feed centrifuges when processing shear-sensitive materials such as the protein precipitates studied here.

The development of Francis Galton's ideas on the mechanism of heredity.

Galton greeted Darwin's theory of pangenesis with enthusiasm, and tried to test the assumption that the heredity particles circulate in the blood by transfusion experiments on rabbits. The failure of these experiments led him to reject this assumption, and in the 1870s he developed an alternative theory of heredity, which incorporated those parts of Darwin's theory that did not involve the transportation of hereditary particles throughout the system. He supposed that the fertilized ovum contains a large number of hereditary elements, which he collectively called the "stirp," a few of which are patent, developing into particular cell types, while the rest remain latent; the latent elements can be transmitted to the next generation, while the patent elements, with rare exceptions, cannot since they have developed into cells. The problem with this theory is that it does not explain the similarity between parent and child unless there is a high correlation between latent and patent elements. Galton probably came to realize this problem during his subsequent statistical work on heredity, and he quietly dropped the idea that patent elements are not transmitted in Natural Inheritance (1889). Galton thought that brothers and sisters had identical stirps, and he attributed differences between them to variability in the choice of patent elements from the stirp, that is to say to developmental variability. He attributed the likeness of monozygotic twins to the similarity of their developmental environment. Galton's twin method was to track the life history changes of twins to see whether twins who were similar at birth diverged in dissimilar environments or whether twins who were dissimilar at birth converged in similar environments. It is quite different from the modern twin method of comparing the similarities between monozygotic and dizygotic twins, on the assumption that monozygotic twins are genetically identical whereas dizygotic twins are not. It has been argued that Galton foreshadowed Weismann's theory in the continuity of the germ-plasm, but this is only true in a weak sense. They both believed that the inheritance of acquired characters was either rare or impossible, but Galton did not forestall the essential part of Weismann's theory, that the germ-plasm of the zygote is doubled, with one part being reserved for the formation of the germ-cells.

Galton's law of ancestral heredity.

Galton's ancestral law states that the two parent contribute between them on average one-half of the total heritage of the offspring, the four grandparents one-quarter, and so on. He interpreted this law both as a representation of the separate contributions of each ancestor to the heritage of the offspring and as a multiple regression formula for predicting the value of a trait from ancestral values. Logical reconstruction of the law is presented based on formalizing Galton's model of heredity outlined in Natural Inheritance (Galton, 1889). The resulting law has a free parameter to be empirically estimated which represents the frequency of latent hereditary elements that are not expressed in a particular individual but are capable of transmission to the next generation. The equation representing ancestral contributions to the heritage of the offspring differs from the multiple regression equation for predicting the value of a trait from ancestral values. The former equation reduces to Galton's ancestral law when the proportion of latent elements is 0.5, the latter when this proportion 0.6. Galton's rather different derivations of the law in 1885 and 1897 are described, and their shortcomings are discussed in the light of these results (Galton, 1885, 1897).

The development of a provincial radiation oncology service: the first year report and its implications for future developments.

This study compares the actual first year's workload of a new radiation oncology department with that predicted, and assesses the impact of the differences, and their implications for future similar developments. The treatment records and diaries for the Geelong Hospital Radiation Oncology Department were reviewed after the first 12 months of operation (opened in June 1992). Statistics relating to the number of patients seen, number treated, diagnosis, etc., were evaluated and compared to the original estimates based upon population statistics and likely referral rates. Nine hundred and seven new patients were seen in this period, and from them 718 courses of treatment were initiated. One hundred and eighty-nine cases (20% of referrals) were seen but not treated. A further 102 treatment courses (14% of total) were initiated upon patients who had previously been irradiated. Forty-six per cent of patients were managed by 10 fractions or fewer, and a further 23% by 25 or more fractions. Eighty per cent of patients were managed by one or two fields. Electrons were used in only 14% of cases. Further calculations suggested a further possible 441 cases of referral could be expected, based upon current population statistics. Referral rates for radiation oncology are highly dependent on a number of factors. As a result, estimates of referrals and hence the size of a department required for a given population vary widely. Our data support these concepts, and the concept that referral is also strongly dependent upon the distance patients need to travel. In considering the development of units outside of major cities it is suggested that referrals are likely to be on the high side of estimates and a minimum two-machine unit is essential to cover the given workload.

Synonymous substitution rates in enterobacteria.

It has been shown previously that the synonymous substitution rate between Escherichia coli and Salmonella typhimurium is lower in highly than in weakly expressed genes, and it has been suggested that this is due to stronger selection for translational efficiency in highly expressed genes as reflected in their greater codon usage bias. This hypothesis is tested here by comparing the substitution rate in codon families with different patterns of synonymous codon use. It is shown that the decline in the substitution rate across expression levels is as great for codon families that do not appear to be subject to selection for translational efficiency as for those that are. This implies that selection on translational efficiency is not responsible for the decline in the substitution rate across genes. It is argued that the most likely explanation for this decline is a decrease in the mutation rate. It is also shown that a simple evolutionary model in which synonymous codon use is determined by a balance between mutation, selection for an optimal codon, and genetic drift predicts that selection should have little effect on the substitution rate in the present case.

A visual programming environment for bioprocess control.

The paper introduces the use of a visual programming environment (LabVIEW) to program custom control functions for bioprocess research. The time taken for a bioprocess scientist to program new functions compared well with typical times expected for experienced programmers using conventional languages. Experienced LabVIEW programmers will develop applications significantly faster. The package described was flexible, easy to use and was ideally suited to developing new applications for control of bioprocesses. It was demonstrated with the development of a system to control specific growth rate in a fed-batch culture.

Reduced synonymous substitution rate at the start of enterobacterial genes.

Synonymous codon usage is less biased at the start of Escherichia coli genes than elsewhere. The rate of synonymous substitution between E.coli and Salmonella typhimurium is substantially reduced near the start of the gene, which suggests the presence of an additional selection pressure which competes with the selection for codons which are most rapidly translated. Possible competing sources of selection are the presence of secondary ribosome binding sites downstream from the start codon, the avoidance of mRNA secondary structure near the start of the gene and the use of sub-optimal codons to regulate gene expression. We provide evidence against the last of these possibilities. We also show that there is a decrease in the frequency of A, and an increase in the frequency of G along the E.coli genes at all three codon positions. We argue that these results are most consistent with selection to avoid mRNA secondary structure.

Cloning and expression of heparinase I gene from Flavobacterium heparinum.

Heparinases, enzymes that cleave heparin and heparin sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies. We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR. Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. This probe was used to screen a Flavobacterium genomic DNA library in pUC18. The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa. Eleven of the tryptic peptides (approximately 35% of the total amino acids) mapped onto the open reading frame. The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site. Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system. The recombinant heparinase cleavage of heparin was identical to that of native heparinase.

The selection-mutation-drift theory of synonymous codon usage.

It is argued that the bias in synonymous codon usage observed in unicellular organisms is due to a balance between the forces of selection and mutation in a finite population, with greater bias in highly expressed genes reflecting stronger selection for efficiency of translation. A population genetic model is developed taking into account population size and selective differences between synonymous codons. A biochemical model is then developed to predict the magnitude of selective differences between synonymous codons in unicellular organisms in which growth rate (or possibly growth yield) can be equated with fitness. Selection can arise from differences in either the speed or the accuracy of translation. A model for the effect of speed of translation on fitness is considered in detail, a similar model for accuracy more briefly. The model is successful in predicting a difference in the degree of bias at the beginning than in the rest of the gene under some circumstances, as observed in Escherichia coli, but grossly overestimates the amount of bias expected. Possible reasons for this discrepancy are discussed.

Strand symmetry of mutation rates in the beta-globin region.

It has been suggested that there may be inequalities in the types of substitution on the two DNA strands (in particular, in the frequencies of transversions from R to Y and from Y to R) due to a higher error rate on the lagging than the leading strand during replication. Reexamination of 11 kb of the beta-globin region sequenced in six primates fails to confirm this suggestion. Examination of the 73-kb beta-globin region sequenced in humans shows that the frequency of pyrimidines in different parts of this region is more variable than expected in a random sequence, but the pattern is more consistent with nonrandomness generated by DNA turnover mechanisms than with strand asymmetry due to a higher error rate on the lagging strand.