Estimation of evolutionary distances between nucleotide sequences

A formal mathematical analysis of the substitution process in nucleotide sequence evolution was done in terms of the Markov process. By using matrix algebra theory, the theoretical foundation of Barry and Hartigan's (Stat. Sci. 2:191–210, 1987) and Lanave et al.'s (J. Mol. Evol. 20:86–93, 1984) methods was provided. Extensive computer simulation was used to compare the accuracy and effectiveness of various methods for estimating the evolutionary distance between two nucleotide sequences. It was shown that the multiparameter methods of Lanave et al.'s (J. Mol. Evol. 20:86–93, 1984), Gojobori et al.'s (J. Mol. Evol. 18:414–422, 1982), and Barry and Hartigan's (Stat. Sci. 2:191–210, 1987) are preferable to others for the purpose of phylogenetic analysis when the sequences are long. However, when sequences are short and the evolutionary distance is large, Tajima and Nei's (Mol. Biol. Evol. 1:269–285, 1984) method is superior to others.     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

Molecules and Morphology,Phylogenetics and Genetics

Various explanations can be offered for the incongruence between phylogenetic hypotheses resulting from morphological and molecular data sets. Of these, the possibility that incongruence may result from the mutation of major morphogenetic genes leading to dramatic morphological divergence unaccompanied by equivalent change of the phylogenetic marker molecule(s) used is discussed in detail. As evidence for this hypothesis, several examples for such incongruence are surveyed. It seems possible that in many cases the genetic basis of the morphological characters responsible for the incongruence found may be simple, and that the genes involved may be homologous to genes known from mutant systems. It is suggested that: 1. the systematic documentation of incongruence between molecular and morphological phylogenies may help to assess the frequency of evolutionary change through the mutation of major morphogenetic genes, and that 2. the identification of major morphological characters distinguishing closely related taxa with mutant phenotypes known from mutant systems eventually may allow an experimental approach to the problem of evolutionary change resulting from major genes. Natural taxa suspected to be the result of such processes could be changed morphologically through transformation with the relevant genes.     

Extractive separation of penicillin G by facilitated transport via carrier supported liquid membranes

The facilitated transport of penicillin G (Pen G), through a supported liquid membrane with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, were studied. The distribution coefficient was obtained from a batch extraction experiment. The effects of flow rate, carrier concentration, initial concentration of Pen G, and the pH of feed and stripping phases on the transport rate of Pen G through the supported liquid membrane were also investigated. The results are in agreement with theoretical predictions, and it is demonstrated that the transport of Pen G through the supported liquid membrane is controlled simultaneously by mass transfer across both aqueous and liquid membranes. (c) 1993 John Wiley & Sons, Inc.     

On the probability of loss of new mutations in the presence of linkage disequilibrium

A new selectively neutral mutation occurs in a multilocus genetic background that has achieved a stable equilibrium at which there is a linkage disequilibrium. Perturbation techniques are applied to an extension of the branching process formulation of Fisher in order to address the question of extinction probabilities. We show that under appropriate conditions the probability of extinction of the new mutant is increased by the existence of linkage disequilibrium in the genetic background.Research supported in part by NIH grant GM 28016     

Rates of decay for the survival probability of a mutant gene II The multitype case

This paper extends the results of [1] to the multitype case. For a multitype branching process that is slightly supercritical, approximations for the survival probability in terms of the maximal eigenvalue of the mean matrix and a generalized variance ² are developed. Our results improve upon those of Hoppe [5] and Eshel [3] that seek to validate a conjecture of Ewens [4].Research supported in part by NSF grant DMS 9007182     

Experimental evidence of size-selective harvest and environmental stochasticity effects on population demography,fluctuations and non-linearity

Theory and analyses of fisheries data sets indicate that harvesting can alter population structure and destabilise non-linear processes, which increases population fluctuations. We conducted a factorial experiment on the population dynamics of Daphnia magna in relation to size-selective harvesting and stochasticity of food supply. Harvesting and stochasticity treatments both increased population fluctuations. Timeseries analysis indicated that fluctuations in control populations were non-linear, and non-linearity increased substantially in response to harvesting. Both harvesting and stochasticity induced population juvenescence, but harvesting did so via the depletion of adults, whereas stochasticity increased the abundance of juveniles. A fitted fisheries model indicated that harvesting shifted populations towards higher reproductive rates and larger-magnitude damped oscillations that amplify demographic noise. These findings provide experimental evidence that harvesting increases the non-linearity of population fluctuations and that both harvesting and stochasticity increase population variability and juvenescence.     

Conformity in mate choice,the overlooked social component of animal and human culture

Although conformity as a major driver for human cultural evolution is a well-accepted and intensely studied phenomenon, its importance for non-human animal culture has been largely overlooked until recently. This limited for decades the possibility of studying the roots of human culture. Here, we provide a historical review of the study of conformity in both humans and non-human animals. We identify gaps in knowledge and propose an evolutionary route towards the sophisticated cultural processes that characterize humanity. A landmark in the study of conformity is Solomon Asch's famous experiment on humans in 1955. By contrast, interest in conformity among evolutionary biologists has only become salient since the turn of the new millennium. A striking result of our review is that, although studies of conformity have examined many biological contexts, only one looked at mate choice. This is surprising because mate choice is probably the only context in which conformity has self-reinforcing advantages across generations. Within a metapopulation, i.e. a group of subpopulations connected by dispersing individuals, dispersers able to conform to the local preference for a given type of mate have a strong and multigenerational fitness advantage. This is because once females within one subpopulation locally show a bias for one type of males, immigrant females who do not conform to the local trend have sons, grandsons, etc. of the non-preferred phenotype, which negatively and cumulatively affects fitness over generations in a process reminiscent of the Fisher runaway process. This led us to suggest a sex-driven origin of conformity, indicating a possible evolutionary route towards animal and human culture that is rooted in the basic, and thus ancient, social constraints acting on mating preferences within a metapopulation. In a generic model, we show that dispersal among subpopulations within a metapopulation can effectively maintain independent Fisher runaway processes within subpopulations, while favouring the evolution of social learning and conformity at the metapopulation scale; both being essential for the evolution of long-lasting local traditions. The proposed evolutionary route to social learning and conformity casts surprising light on one of the major processes that much later participated in making us human. We further highlight several research avenues to define the spectrum of conformity better, and to account for its complexity. Future studies of conformity should incorporate experimental manipulation of group majority. We also encourage the study of potential links between conformity and mate copying, animal aggregations, and collective actions. Moreover, validation of the sex-driven origin of conformity will rest on the capacity of human and evolutionary sciences to investigate jointly the origin of social learning and conformity. This constitutes a stimulating common agenda and militates for a rapprochement between these two currently largely independent research areas.     

Reaction intensification for biocatalytic production of polyphenolic natural product di-C-β-glucosides

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.