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Overview of screening for new microbial catalysts and their uses in organic synthesis--selection and optimization of biocatalysts.

As a typical example of screening for a microbial biocatalyst from nature, isolation of aldoxime-degrading microorganisms, characterization of a new enzyme phenylacetaldoxime dehydratase, and application of this enzyme to nitrile synthesis are described. The pathway in which aldoximes are successively degraded via nitrile in microorganisms could be named as 'aldoxime-nitrile pathway'. As an example of a post-screening procedure, a directed molecular evolution technique was successfully used to change the properties of nucleoside pyrophosphate phosphotransferase to make it suitable for synthesis of inosine-5'-monophosphate (5'-IMP). With the mutant enzyme, the efficiency of the production of 5'-IMP, a food additive, was much improved.

Biodegradation, Environmental↗

How to broaden enzyme substrate specificity: strategies, implications and applications.

For identification of mutations associated with the broadening of enzyme substrate specificity, three strategies, including directed enzyme evolution, are described for selected examples. Implications concerning enzyme models are highlighted. Applications to the field of biocatalysis are discussed. A bidimensional map for the classification of enzyme activities is suggested so as to improve genome annotations.

Catalysis↗

Dispelling the myths--biocatalysis in industrial synthesis.

Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.

Biotechnology↗

[The characteristics of the evolutionary variability of influenza A (H1N1) viruses].

Studies of the antigenic structure of hemagglutinins of influenza A (H1N1) viruses isolated in 1978-1988 using monospecific and monoclonal antibodies demonstrated the strains of the H1N1 subtype to be highly apt to antigenic drift. The evolutional variability of that period was peculiar and characterized by antigenic drift in various directions. In those years, the variants were regularly isolated which had retained the determinants of viruses of 1933-1957 circulation period in their hemagglutinin structure. The variants containing in their hemagglutinin 2 antigenic sites common with A/USSR/090/77 virus and antigenic groupings characterizing the strain specificity of each isolate, were epidemically active. At the same time, epidemically important variants were dominant whose properties were markedly different from those of previously known viruses. Their hemagglutinin contained 2 basically new antigenic determinants. This direction of evolutional development of influenza A (H1N1) virus is the most prospective epidemically.

Animals↗

Overcoming antigenic diversity and improving vaccines using DNA shuffling and screening technologies.

Viral, bacterial and parasitic pathogens have evolved multiple strategies to evade the immune response, facilitate transmission and establish chronic infections. One of the underlying strategies that pathogens have evolved is antigenic variation of immune response targets that reduce the affinity of antigen binding to antibodies and major histocompatability complex class I and II receptors. Vaccine candidates generally target a limited number of these antigen variants or combine antigens from several variants to include in multivalent vaccine formulations. DNA shuffling and screening technologies, also known as MolecularBreeding (Maxygen, Inc.) directed molecular evolution, have been successfully used to identify and develop novel and chimaeric vaccine candidates capable of inducing immune responses that recognise and control multiple antigenic variants. DNA shuffling and screening strategies also select vaccine candidates with improved immunogenicity, increased expression as recombinant polypeptides and improved growth of whole viruses in cell culture. As DNA shuffling and screening strategies can be applied to many pathogens, there remain numerous applications of DNA shuffling to solve challenging problems in vaccine process development and manufacture.

Animals↗

Climate variability and the energetic pathways of evolution: the origin of endothermy in mammals and birds.

Large-scale climate oscillations in earth's history have influenced the directions of evolution, last but not least, through mass extinction events. This analysis tries to identify some unifying forces behind the course of evolution that favored an increase in organismic complexity and performance, paralleled by an increase in energy turnover, and finally led to endothermy. The analysis builds on the recent concept of oxygen-limited thermal tolerance and on the hypothesis that unifying principles exist in the temperature-dependent biochemical design of the eukaryotic cell in animals. The comparison of extant water-breathing and air-breathing animal species from various climates provides a cause-and-effect understanding of the trade-offs and constraints in thermal adaptation and their energetic consequences. It is hypothesized that the high costs of functional adaptation to fluctuating temperatures, especially in the cold (cold eurythermy), cause an increase in energy turnover and, at the same time, mobility and agility. These costs are associated with elevated mitochondrial capacities at minimized levels of activation enthalpies for proton leakage. Cold eurythermy is seen as a precondition for the survival of evolutionary crises elicited by repeated cooling events during extreme climate fluctuations. The costs of cold eurythermy appear as the single most important reason why metazoan evolution led to life forms with high energy turnover. They also explain why dinosaurs were able to live in subpolar climates. Finally, they give insight into the pathways, benefits, and trade-offs involved in the evolution of constant, elevated body temperature maintained by endothermy. Eurythermy, which encompasses cold tolerance, is thus hypothesized to be the "missing link" between ectothermy and endothermy. Body temperatures between 32 degrees and 42 degrees C in mammals and birds then result from trade-offs between the limiting capacities of ventilation and circulation and the evolutionary trend to maximize performance at the warm end of the thermal tolerance window.

Adaptation, Physiological↗

Quantitative genetics and developmental constraints on evolution by selection.

It has often been argued that the principles of random mutation and selection are insufficient to account for macroevolutionary phenomena, such as the origin of morphological novelty and directionality in evolution. A third, epigenetic, principle is said to be required and this principle is thought not to be included in microevolutionary theory. The third principle has most recently been identified as internal selection and/or non-random phenotypic effects of mutation. It is shown that the genetic variance/covariance matrix of quantitative genetic theory measures developmental constraints due to internal selection and non-random mutation. The genetic variance/covariance matrix causes the response to selection to deviate from the optimal rate and direction as specified by the selection gradient, which measures direct selection on the phenotypes. Therefore, microevolutionary theory takes account of developmental constraints on evolution by natural selection through the genetic variance/covariance matrix. Theories for predicting the pattern of genetic variance and covariance from stabilizing selection and the phenotypic effects of mutation are discussed.

Biological Evolution↗

Cranial neural crest-cell migration in the direct-developing frog, Eleutherodactylus coqui: molecular heterogeneity within and among migratory streams.

Direct development is a specialized reproductive mode that has evolved repeatedly in many different lineages of amphibians, especially anurans. A fully formed, albeit miniature adult hatches directly from the egg; there is no free-living larva. In many groups, the evolution of direct development has had profound consequences for cranial development and morphology, including many components that are derived from the embryonic neural crest. Yet, the developmental bases of these effects remain poorly known. In order to more fully characterize these changes, we used three molecular markers to analyze cranial neural crest-cell emergence and migration in the direct-developing frog, Eleutherodactylus coqui: HNK-1 immunoreactivity, Dlx protein expression, and cholinesterase activity. Our study validates and extends earlier results showing that the comprehensive changes in embryonic cranial patterning, differentiation, and developmental timing that are associated with direct development in Eleutherodactylus have not affected gross features of cranial neural crest biology: the relative timing of crest emergence and the number, configuration and identity of the principal migratory streams closely resemble those seen in metamorphic anurans. The three markers are variably expressed within and among neural crest-cell populations. This variation suggests that determination of cranial neural crest-cells may already have begun at or soon after the onset of migration, when the cells emerge from the neural tube. It is not known how or even if this variation correlates with differential cell lineage or fate. Finally, although HNK-1 expression is widely used to study neural crest migration in teleost fishes and amniotes, E. coqui is the only amphibian known in which it effectively labels migrating neural crest-cells. There are not enough comparative data to determine whether this feature is functionally associated with direct development or is instead unrelated to reproductive mode.

Journal Article↗

Development of novel vaccines using DNA shuffling and screening strategies.

DNA shuffling and screening technologies recombine and evolve genes in vitro to rapidly obtain molecules with improved biological activity and fitness. In this way, genes from related strains are bred like plants or livestock and their successive progeny are selected. These technologies have also been called molecular breeding-directed molecular evolution. Recent developments in bioinformatics-assisted computer programs have facilitated the design, synthesis and analysis of DNA shuffled libraries of chimeric molecules. New applications in vaccine development are among the key features of DNA shuffling and screening technologies because genes from several strains or antigenic variants of pathogens can be recombined to create novel molecules capable of inducing immune responses that protect against infections by multiple strains of pathogens. In addition, molecules such as co-stimulatory molecules and cytokines have been evolved to have improved T-cell proliferation and cytokine production compared with the wild-type human molecules. These molecules can be used to immunomodulate vaccine responsiveness and have multiple applications in infectious diseases, cancer, allergy and autoimmunity. Moreover, DNA shuffling and screening technologies can facilitate process development of vaccine manufacturing through increased expression of recombinant polypeptides and viruses. Therefore, DNA shuffling and screening technologies can overcome some of the challenges that vaccine development currently faces.

Animals↗

DNA shuffling and screening strategies for improving vaccine efficacy.

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.

Antigens↗

Aligned 18S and insect phylogeny.

The nuclear small subunit rRNA (18S) has played a dominant role in the estimation of relationships among insect orders from molecular data. In previous studies, 18S sequences have been aligned by unadjusted automated approaches (computer alignments that are not manually readjusted), most recently with direct optimization (simultaneous alignment and tree building using a program called "POY"). Parsimony has been the principal optimality criterion. Given the problems associated with the alignment of rRNA, and the recent availability of the doublet model for the analysis of covarying sites using Bayesian MCMC analysis, a different approach is called for in the analysis of these data. In this paper, nucleotide sequence data from the 18S small subunit rRNA gene of insects are aligned manually with reference to secondary structure, and analyzed under Bayesian phylogenetic methods with both GTR+I+G and doublet models in MrBayes. A credible phylogeny of Insecta is recovered that is independent of the morphological data and (unlike many other analyses of 18S in insects) not contradictory to traditional ideas of insect ordinal relationships based on morphology. Hexapoda, including Collembola, are monophyletic. Paraneoptera are the sister taxon to a monophyletic Holometabola but weakly supported. Ephemeroptera are supported as the sister taxon of Neoptera, and this result is interpreted with respect to the evolution of direct sperm transfer and the evolution of flight. Many other relationships are well-supported but several taxa remain problematic, e.g., there is virtually no support for relationships among orthopteroid orders. A website is made available that provides aligned 18S data in formats that include structural symbols and Nexus formats.

Animals↗

Analysis of directional mutation pressure and nucleotide content in mitochondrial cytochrome b genes.

We present a new approach for analyzing directional mutation pressure and nucleotide content in protein-coding genes. Directional mutation pressure, the heterogenicity in the likelihood of different nucleotide substitutions, is used to explain the increasing or decreasing guanine-cytosine content (GC%) in DNA and is represented by microD, in agreement with Sueoka (1962, Proc Natl Acad Sci USA 48:582-592). The new method uses simulation to facilitate identification of significant A+T or G+C pressure as well as the comparison of directional mutation pressure among genes, even when they are translated by different genetic codes. We use the method to analyze the evolution of directional mutation pressure and nucleotide content of mitochondrial cytochrome b genes. Results from a survey of 110 taxa indicate that the cytochrome b genes of most taxa are subjected to significant directional mutation pressure and that the gene is subject to A+T pressure in most cases. Only in the anseriform bird Cairina moschata is the cytochrome b gene subject to significant G+C pressure. The GC% at nonsynonymous codon sites decreases proportionately with increasing A+T pressure, and with a slope less than one, indicating a presence of selective constraints. The cytochrome b genes of insects, nematodes, and eumycotes are subject to extreme A+T pressures (microD = 0.123, 0.224, and 0.130) and, in parallel, the GC% of the nonsynonymous codon sites has decreased from about 0.44 in organisms that are not subjected to A+T or G+C pressure to about 0.332, 0.323, and 0.367, respectively. The distribution of taxa according to the GC% at nonsynonymous codon sites and directional mutation pressure supports the notion that variation in these parameters is a phylogenetic component.

Biological Evolution↗

Experimental evolution of conflict mediation between genomes.

Transitions to new levels of biological complexity often require cooperation among component individuals, but individual selection among those components may favor a selfishness that thwarts the evolution of cooperation. Biological systems with elements of cooperation and conflict are especially challenging to understand because the very direction of evolution is indeterminate and cannot be predicted without knowing which types of selfish mutations and interactions can arise. Here, we investigated the evolution of two bacteriophages (f1 and IKe) experimentally forced to obey a life cycle with elements of cooperation and conflict, whose outcome could have ranged from extinction of the population (due to selection of selfish elements) to extreme cooperation. Our results show the de novo evolution of a conflict mediation system that facilitates cooperation. Specifically, the two phages evolved to copackage their genomes into one protein coat, ensuring cotransmission with each other and virtually eliminating conflict. Thereafter, IKe evolved such extreme genome reduction that it lost the ability to make its own virions independent of f1. Our results parallel a variety of conflict mediation mechanisms existing in nature: evolution of reduced genomes in symbionts, cotransmission of partners, and obligate coexistence between cooperating species.

Bacteriophages↗

[New concepts in the approach to congenital strabismus].

The first part of the paper reviews the clinical symptoms of congenital strabismus. Congenital strabismus passes four stages during its evolution. Spontaneous evolution becomes either alternant strabismus (stage IV a) or monolateral strabismus (stage IV b). The early treatment is to direct the evolution to the "a" form of stages III and IV and mainly consists of optical correction and occlusion.

Fixation, Ocular↗

Evolution and current status of direct-entry midwifery education, regulation, and practice in the United States, with examples from Washington State.

This paper describes the re-emergence of direct-entry midwifery in the United States, and focuses specifically on the over 1,000 midwives nationwide who are licensed in the 16 states where direct-entry midwifery is legal and regulated, and/or certified by the North American Registry of Midwives; it does not focus on direct-entry midwives or nurse-midwives who are certified by the American College of Nurse-Midwives Certification Council, Inc. Professional developments of direct-entry midwives are highlighted, including the establishment of core competencies and articulation of values, the creation of a certification process, and development of education program accreditation. The current status of licensed midwives in Washington State, where state policies have supported the development of direct-entry midwifery and the integration of direct-entry midwives into managed care systems, is presented as one example of the evolution of professional direct-entry midwifery in this country. Additionally, recommendations from the UCSF Center for the Health Professions Taskforce on Midwifery, which address particular areas of concern for direct-entry midwives, are discussed.

Certification↗

Facial nerve surgery in the 19th and early 20th centuries: The evolution from crossover anastomosis to direct nerve repair.

The historical aspects of facial nerve (FN) anatomy and of Bell's palsy have long been favorite topics of otologic historians. Little attention has been paid, however, to the evolution of FN surgery, a subject with a remarkably rich and engaging history. In the early 13th century, Roland, an Italian surgeon, used a red hot iron to coapt severed nerve endings. In the 17th century, Ferrara, another Italian, sutured injured nerves with tortoise tendon dipped in hot red wine. It was not until the late 19th century that peripheral nerve suture became a subject of serious scientific study. Although it is ironic, the course of events suggests that the evolution of FN repair was greatly stimulated by the development of the modern mastoid operation. Whereas the simple mastoid operation practiced by Wilde (1853) and others carried little risk of FN injury, more adventuresome procedures such as radical mastoidectomy (Kessel, 1885) carried a much greater risk. The abundance of iatrogenic palsies during this era undoubtedly did much to motivate surgeons to seek a better means of restoring facial animation. Most surgeons would be surprised to learn that crossover anastomoses predated direct nerve repair by nearly half a century. In 1879, the German surgeon Drobnik performed the first facial-spinal accessory anastomosis. Over the next two decades, numerous articles were written (most notably by Sir Charles Balance and Harvey Cushing) on crossovers between the FN and cranial nerves IX, X, XI, and XII. Although a few tentative attempts at reapproximating severed FNs took place in the first two decades of this century, it was not until 1925 that an actual suture repair of an intratemporal injury was undertaken. This feat was first accomplished by the famous hand surgeon Sterling Bunnell and shortly thereafter by the otolaryngologist Robert Martin. The evolution of FN surgery in the days predating the operating microscope is a rich tapestry of colorful personalities and clashing egos, which saw promising advances relegated to obscurity and some previously obscure techniques become progressively more promising.

Anastomosis, Surgical↗

Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry.

Direct development in amphibians is an evolutionarily derived life-history mode that involves the loss of the free-living, aquatic larval stage. We examined embryos of the direct-developing anuran Eleutherodactylus coqui (Leptodactylidae) to evaluate how the biphasic pattern of cranial ontogeny of metamorphosing species has been modified in the evolution of direct development in this lineage. We employed whole-mount immunohistochemistry using a monoclonal antibody against the extracellular matrix component Type II collagen, which allows visualization of the morphology of cartilages earlier and more effectively than traditional histological procedures; these latter procedures were also used where appropriate. This represents the first time that initial chondrogenic stages of cranial development of any vertebrate have been depicted in whole-mounts. Many cranial cartilages typical of larval anurans, e.g., suprarostrals, cornua trabeculae, never form in Eleutherodactylus coqui. Consequently, many regions of the skull assume an adult, or postmetamorphic, morphology from the inception of their development. Other components, e.g., the lower jaw, jaw suspensorium, and the hyobranchial skeleton, initially assume a mid-metamorphic configuration, which is subsequently remodeled before hatching. Thirteen of the adult complement of 17 bones form in the embryo, beginning with two bones of the jaw and jaw suspensorium, the angulosplenial and squamosal. Precocious ossification of these and other jaw elements is an evolutionarily derived feature not found in metamorphosing anurans, but shared with some direct-developing caecilians. Thus, in Eleutherodactylus cranial development involves both recapitulation and repatterning of the ancestral metamorphic ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The evolution of senescence in natural populations of guppies (Poecilia reticulata): a comparative approach.

Model organisms like Drosophila melanogaster or Caenorhabditis elegans have revealed genes that influence senescence and the evolvability of senescence. We are interested instead in evaluating why and how senescence evolves in natural populations. To do so, we are taking the ecological geneticist's perspective of comparing natural populations that differ in factors that are predicted to influence the evolution of senescence and are evaluating whether senescence has evolved in the predicted fashion. We are also manipulating the environment to evaluate more directly the evolution of senescence. Guppies (Poecilia reticulata) are found in streams throughout the Northern Range mountains of Trinidad. Natural populations experience large differences in mortality rate as a consequence of the predators with which they co-occur. We have already shown, both with comparative studies and manipulations of the distribution of guppies and their predators, that the early life history evolves very rapidly in response to these differences in mortality. For example, high adult mortality rates select for individuals that develop more rapidly, produce their first litter of young at an earlier age, and devote more of their available resources to reproduction for the remainder of their lives. These changes were predicted by independently derived theory. Aspects of this same theory also predict how the late life history and senescence should evolve. Specifically, theory predicts that the populations that experience low mortality rates should also experience delayed senescence and longer life spans relative to those that experience high mortality rates. We are currently evaluating these predictions with representatives from two high-predation and two low-predation environments. Our presentation will focus on our pilot study, which evaluated life span, lifetime reproduction, and the patterns of aging in our laboratory populations. We will also report on the progress in our ongoing comparative studies of senescence in natural populations.

Aging↗