Question 9: Prospects for the Construction of Artificial Cells or Protocells

The construction of artificial cells or protocells that are a simplified version of contemporary cells will have implications for both the understanding of the origins of cellular Life and the design of “cell-like” chemical factories. In this short communication, we discuss the progress and remaining issues related to the construction of protocells from metabolic products. We further outline the de novo design of a simple chemical system that mimics the functional properties of a living cell without being composed of molecules of biological origin, thereby addressing issues related to Life’s origins. Presented at: International School of Complexity—4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Question 9: Quantum Self-Assembly and Photoinduced Electron Tunneling in Photosynthetic Systems of Artificial Minimal Living Cells

Natural and artificial living cells and their substructures are self-assembling, due to electron correlation interactions among biological and water molecules, which lead to attractive dispersion forces and hydrogen bonds. Dispersion forces are weak intermolecular forces that arise from the attractive force between quantum multipoles. A hydrogen bond is a special type of quantum attractive interaction that exists between an electronegative atom and a hydrogen atom bonded to another electronegative atom; and this hydrogen atom exist in two quantum states. The best method to simulate these dispersion forces and hydrogen bonds is to perform quantum mechanical non-local density functional potential calculations of artificial minimal living cells consisting of around 1,000 atoms. The cell systems studied are based on peptide nucleic acid and are 3.0–4.2 nm in diameter. The electron tunneling and associated light absorption of the most intense transitions, as calculated by the time dependent density functional theory method, differs from spectroscopic experiments by only 0.2–0.3 nm, which is within the value of experiment errors. This agreement implies that the quantum mechanically self-assembled structures of artificial minimal living cells very closely approximate realistic ones.     

Question 7: Construction of a Semi-Synthetic Minimal Cell: A Model for Early Living Cells

Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named “Puresystem” (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30–20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Self-organization in dissipative structures: A thermodynamic theory for the emergence of prebiotic cells and their epigenetic evolution

This paper presents a discussion on self-organization processes in dissipative structures, in order to highlight the general conditions for raising complexity and generate order. In particular, some concepts were introduced from non-equilibrium thermodynamics and from the Molecular Anamorphic Evolution Theory, especially concerning processes of matter randomization.     

Evaluation of the Predatory Wasp, Ancistrocerus gazella, for Biological Control of Leafrollers in Otago Fruit Crops: 1. Prey Composition, Nest Structure and Wasp Productivity from Artificial Nests

Groups of artificial nest tubes were deployed in the field for colonization by the accidentallyimported wasp, Ancistrocerus gazella (Panzer), a reported important predator of lepidopterous larvae, especially pest tortricids. There were 3-4 nest sites in each of seven study areas, and four tube lengths (75, 100, 125, 150 mm) were compared. Nests were dissected at various stages after occupancy to determine the prey composition and nest structure, and the numbers and sex of adults produced. The wasps collected larvae of nine lepidopterous families, including many tortricids, but pest leafrollers comprised only 4.1% and 1.5% of all larvae collected respectively in 1995-96 and 1996-97. Prey included beneficial species feeding on weeds and the larvae of some non-pest native insects. Prey species composition was highly site-dependent and was a major determinant of the numbers of larvae per cell. Cells for male wasps were smaller and contained fewer larvae than those for females. Tube length did not affect colonization rates but more cells and adult wasps were produced in longer tubes. The results cast serious doubt on the proposed management of A. gazella for biological control of leafrollers, but suggest that this approach could be useful for other pests, such as Coleophora spp., which were collected in large numbers.     

On the Evolution of Feathers from an Aerodynamic and Constructional View Point

The evolution of birds and feathers are examined in terms ofthe aerodynamic constraints imposed by the arboreal and cursorialmodels of flight evolution. The cursorial origin of flight isassociated with the putative coelurosaurian ancestry of birds.As presently known, coelurosaurs have a center of mass locatedin the pelvic region and an elongated pubis that is ventrallyor anteriorly directed. Both of these characteristics make itdifficult to postulate an origin of flight that would involvea gliding phase because the abdomen cannot be flattened intoan aerodynamic shape. Moreover, the cursorial model must counteractgravity using the hindlimb and, thus, selection for the powerrequirement for lift-off would not focus on the forelimb. Therefore,if the hypothesis proposing a coelurosaurian ancestry of birdsis to remain viable, it must be via an as yet undiscovered taxonthat is compatible with the morphological and aerodynamic constraintsimposed by flight evolution. The arboreal model, currently centers around non-dinosauriantaxa and is more parsimonious in that early archosaurs haveshort pubes that do not preclude an aerodynamic body profile.Moreover, the arboreal proavis uses gravity to create the airflowover the body surfaces and is, thus, energy efficient. Considerationof the initial aerodynamic roles of feathers and feather designare consistent with a precursory gliding phase. Whether avianancestry lies among coelurosaur theropods or earlier archosaurs,we must remain mindful of the complex aerodynamic dictates ofgliding and powered flight and avoid formalistic approachesthat co-opt sister taxa, with their known body form, as functionalancestors.     

Question 1: Origin of Life and the Living State

The aim of this article is to discuss four topics: First, the origin of molecular reproduction. Second, the origin of agency – the capacity of a system to act on its own behalf. Agency is a stunning feature of human and some wider range of life. Third, to discuss a still poorly articulated feature of life noticed by the philosopher Immanuel Kant over 200 years ago: A self propagating organization of process. We have no theory for this aspect of life, yet it is central to life. Fourth, I will discuss constraints, as in Schroedinger’s aperiodic crystal (Schroedinger E, What is life? The physical aspect of the living cell, 1944), as information, part of the total non-equilibrium union of matter, energy, work, work cycles, constraints, and information that appear to comprise the living state.     

Artificial cultivation of true morels: current state,issues and perspectives

Morels (Morchella, Ascomycota), which are some of the most highly prized edible and medicinal mushrooms, are of great economic and scientific value. Morel cultivation has been a research focus worldwide for more than 100 years, and the outdoor cultivation of morels has succeeded and expanded to a large scale in China in recent years. In this study, we review the progress in recent research regarding the life cycle and reproductive systems in the genus Morchella and the current state of outdoor cultivation. Sclerotia formation and conidia production are two important phases during the life cycle. The morel species cultivated commercially in America is M. rufobrunnea based on molecular phylogenetic analysis. The species currently cultivated in China are black morels, including M. importuna, M. sextalata and M. eximia. The field cultivation of morels expanded in the majority of the provinces in China with a yield of fresh morels of 0–7620?kg per ha. The key techniques include spawn production, land preparation and spawning, the addition of exogenous nutrition, fruiting management and harvesting. The application of exogenous nutrition is the most important breakthrough in the field of morel cultivation, but the mechanism remains unclear. It was estimated that the total amount of field cultivated fresh morels was ～500 t in 2015–2016. We also discuss the potential issues remaining in the current literature and suggest directions for future studies.     

Life Cycle Approach in the Procurement Process: The Case of Defence Materiel (9 pp)

Goal, Scope and Background Procurement in public and non-public organisations has the potential to influence product development towards more environmentally friendly products. This article focuses on public procurement with procurement in Swedish defence as a special case. In 2003, public procurement in Sweden was 28% of the GDP. In the Swedish defence sector the amount was 2% of the GDP. The total emissions from the sector were of the same order of magnitude as from waste treatment (2% of Sweden's emissions). According to an appropriation letter from the Ministry of Defence in 1998, the Swedish Armed Forces (SAF) and the Swedish Defence Materiel Administration (FMV) are required to take environmental issues into consideration during the entire process of acquiring defence materiel. Environmental aspects are considered today, but without a life-cycle perspective. - The aims of this article are to recommend suitable tools for taking environmental concerns into account, considering a product's life-cycle, in the procurement process for defence materiel in Sweden; to make suggestions for how these tools could be used in the acquisition process; and to evaluate these suggestions through interviews with actors in the acquisition process. The procurement process does not include aspects specific to Swedish defence, and it is therefore likely to be comparable to processes in other countries. Methods The method involved a study of current literature and interviews with various actors in the acquisition process. The life cycle methods considered were quantitative Life Cycle Assessments, a simplified LCA-method called the MECO method and Life Cycle Costing (LCC). Results and Discussion Methodology recommendations for quantitative LCA and simplified LCA are presented in the article, as well as suggestions on how to integrate LCA methods in the acquisition process. We identified four areas for use for LCA in the acquisition process: to learn about environmental aspects of the product; to fulfil requirements from customers; to set environmental requirements and to choose between alternatives. Therefore, tools such as LCAs are useful in several steps in the acquisition process. Conclusion From the interviews, it became clear that the actors in the acquisition process think that environmental aspects should be included early in the process. The actors are interested in using LCA methods, but there is a need for an initiative from one or several of them if the method is to be used regularly in the process. Environmental and acquisition issues are handled with very little interaction in the controlling and ordering organisation. An integration of environmental and acquisition parts in these organisations is probably needed in order to integrate environmental aspects in general and life-cycle thinking in particular. Other difficulties identified are costs and time constraints. Recommendation and Perspective In order to include the most significant aspects when procuring materiel, it is important to consider the whole life-cycle of the products. Our major recommendation is that the defence sector should work systematically through different product groups. For each product group, quantitative, traditional LCAs or simplified LCAs (in this case modified MECOs) should be performed for reference products within each product group. The results should be an identification of critical aspects in the life-cycles of the products. The studies will also form a database that can be used when making new LCAs. This knowledge should then be used when writing specifications of what to procure and setting criteria for procurement. The reports should be publicly available to allow reviews and discussions of results. To make the work more cost-effective, international co-operation should be sought. In addition, LCAs can also be performed as an integrated part of the acquisition process in specific cases.     

On the Emergence of Biology from Chemistry: A Discontinuist Perspective from the Point of View of Stability and Regulation

In this paper we argue that molecular evolution, and the evolution of prebiotic and early biological systems are qualitatively different processes, in which a crucial role is played respectively by structural stability and by dynamical mechanisms of regulation and integration. These different features entail also distinct modalities of interaction between system and environment that need to be taken into consideration when discussing molecular and biological evolution and selection.     

Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

On the politics and practice of Muslim fertility: comparative evidence from West Africa

Recent popular works have represented Muslim fertility as dangerously high, both a cause and consequence of religious fundamentalism. This article uses comparative, statistical methods to show that this representation is empirically wrong, at least in West Africa. Although religion strongly inflects reproductive practice, its effects are not constant across different communities. In West African countries with Muslim majorities, Muslim fertility is lower than that of their non-Muslim conationals; in countries where Muslims are in the minority, their apparently higher reproductive rates converge to those of the majority when levels of education and urban residence are taken into account. A similar pattern holds for infant mortality. By contrast, in all seven countries, Muslim women are more likely to report that their most recent child was wanted. The article concludes with a discussion of the relationship between autonomy and fertility desires.     

Development of a urinary biomarker for exposure to the organophosphate propetamphos: data from an oral and dermal human volunteer study

Propetamphos is a member of the vinyl phosphate group of insecticides and is mainly used for sheep dipping. There have been no published metabolic studies on the effect of propetamphos in man to date, although the present authors have published the identification of a metabolite. The present paper presents data from a human volunteer study investigating the toxicokinetics of the organophosphorus pesticide propetamphos following oral and dermal exposure. Five volunteers ingested a propetamphos dose of 10 μg kg^-1 (35nmol kg^-1) body weight. Following a washout of 4 weeks, a 100mg (356 μmol) dermal dose of propetamphos was applied, occluded to 80cm² of the inner forearm, for 8 h to the same five volunteers. In a pilot study (several weeks before the main study), one volunteer also received an occluded dermal dose of 50 mg (178 μmol) propetamphos. Unabsorbed propetamphos on the skin was washed off after 8 h and collected. Blood and urine samples were collected over 30 and 54 h for the oral and dermal exposures respectively. Blood samples were analysed for plasma and erythrocyte cholinesterase. Urine samples were analysed for a urinary metabolite of propetamphos: methylethylphosphoramidothioate (MEPT). Following oral and dermal exposure, peak urinary MEPT levels occurred at 1 and 10-12 h respectively. The apparent urinary elimination half-lives of MEPT had means of 1.7h (oral exposure) and 3.8 h (dermal exposure). Approximately 40% of the oral dose and 1% of the dermal dose were recovered as urinary MEPT or metabolites, which could be hydrolysed to MEPT. Approximately 90% of the dermal dose was recovered from the skin washings. Data from a volunteer showed that a doubling of the dermal dose resulted in approximately double the concentration of total MEPT. Alkaline hydrolysis of urine samples increased the level of MEPT detected after both oral and dermal doses. The increase was greater and statistically significant (p < 0.001, paired t-test) for the dermal dose. This increase in MEPT suggests the presence of other MEPT-containing metabolites or conjugates. The difference in the increase between oral and dermal doses raises the question of a difference in metabolism between the two routes. No individual showed a significant depression compared with their pre-exposure levels of erythrocyte acetyl cholinesterase or plasma cholinesterase activity for either dosing route. However, on a group basis, there was a statistically significant mean depression in plasma cholinesterase activity at 8 and 24 h for oral exposure, with a maximum mean depression of 7% from pre-exposure levels at 8 h. Hydrolysis of urine samples had the effect of reducing the interindividual coefficient of variation (CV) for total excretion of MEPT following both oral (CV reduced from 36 to 8%) and dermal (CV reduced from 40 to 17%) exposure. The ability to detect and follow the elimination of low doses of propetamphos by measurement of 'total' (after hydrolysis) urinary MEPT suggests it is a suitable biomarker of propetamphos exposure. The comparatively short elimination half-lives suggest a strategy for biological monitoring of occupational exposure based on samples collected at the end of the shift.     

Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation in E. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.     

Two-sex life table of Nasonia vitripennis (Hymenoptera: Pteromalidae) and the effects of Wolbachia on its reproduction and parasitism of Musca domestica (Diptera: Muscidae)

Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) is now emerging as genetic model organism for development and genetics research. As a parasitic insect, the egg, larval, pupal, and early adult developmental stages of N. vitripennis occur within the enclosed fly pupae, which differ a lot from the life cycle of other insects that undergo complete metamorphosis. Previous report on the life table of N. vitripennis was based on females only. In this study, the two-sex life table approach was used to examine the parasitic efficiency of N. vitripennis within the pupae of Musca domestica L. (Diptera: Muscidae), and the influence of low temperature and the endosymbiotic bacteria Wolbachia on wasp population growth were investigated. Wolbachia could improve the fecundity of N. vitripennis and prolong the host life history. We propose the preservation of M. domestica pupae at 4 °C for 15 days is suitable in practical use. Age-stage, two-sex life table analysis revealed the stage structure and variability of N. vitripennis population growth, and also provide useful information about the effects of Wolbachia on its reproduction and parasitism of M. domestica.     

Artificial diet and rearing methods for Thanasimus dubius (Coleoptera: Cleridae), a predator of bark beetles (Coleoptera: Scolytidae)

Clerid beetles are common natural enemies of bark beetles, and could potentially be used as biological control agents if they could be reared in sufficient numbers. We developed an artificial diet devoid of insect components for rearing Thanasimus dubius (Fabricius), a clerid that attacks several economically important bark beetles in eastern North America. We reared larvae of this predator using the artificial diet, and then used either natural or factitious prey to feed the adults so produced. Two different methods of presenting the diet were also examined. We then compared the performance of T. dubius reared on the artificial diet with newly-emerged wild individuals collected from the field. Our results suggest that adult predators reared on the diet are near in quality to wild ones, and high R₀ values can be obtained. No difference in prey preference was found between wild and diet-reared individuals after five generations in the laboratory. Sufficient numbers of predators could be generated using these techniques to permit limited field trials of augmentative biological control.     

Olencira praegustator (Crustacea, Isopoda, Cymothoidae) parasitic on Brevoortia species (Pisces, Clupeidae) from the southeastern coasts of North America: review and re-description

The cymothoid isopod Olencira praegustator (Latrobe, 1802), the sole species of the genus Olencira Leach, 1818, has been the subject of several previous studies, but has remained incompletely described. This species is re-described and figured from the Atlantic menhaden Brevoortia tyrannus (Latrobe, 1802), the type host from the estuarine systems of the South Carolina coast, USA. The general morphology, mouthparts and appendages of the female, male and other stages of its lifecycle are described.

The geographical distribution and hosts recorded for O. praegustator are reviewed. Biological data are summarized and discussed. Areas that need additional studies include the infection process and the effect of the parasite on its host.     

Biology and host specificity ofChamaesphecia hungarica andCh. Astatiformis (Lep.: Sesiidae) two candidates for the biological control of leafy spurge,Euphorbia esula (Euphorbiaceae) in North America

Leafy spurge (Euphorbia esula (s.1.)) is an herbaceous perennial and serious weed of Eurasian origin that has been accidentally introduced into North America. The two European root-boring mothsChamaesphecia hungarica andCh. astatiformis are univoltine and overwinter as mature larvae. Both species have a lower survival rate on leafy spurge than on their field hosts, and thus are not optimal candidates for the biological control of leafy spurge. However, the rate of larval development and larval growth on the target weed and on the two field hosts is nearly the same. The experimental host range of both species is restricted to a few species in the subgenusEsula within the genusEuphorbia. The two species occupy different habitats in the steppe biome and are targeted for similar leafy spurge habitats in North America.