Performance of the Wheat Armyworm, Pseudaletia sequax Franclemont, on Natural and Artificial Diets

Armyworms in the genus Pseudaletia Franclemont are important pests of cereal crops worldwide. The development of pest management strategies is largely dependent on studies involving the relationship among the pest, its host plants and natural enemies, as well as its susceptibility to chemical and biological control strategies. Part of these studies requires rearing the species of interest under laboratory conditions, which can be greatly facilitated by the use of artificial diets. In the present study, the effects of three artificial diets on development, survival, reproduction and demographic parameters of P. sequax were compared to a natural host plant, Pennisetum clandestinum. A diet based on pinto beans, wheat germ and torula yeast provided the best results in comparison to those obtained with the host plant. The most important life history parameters used in the evaluation of the nutritional quality of the diets were fecundity, egg viability and pupae deformities. The intrinsic rate of increase and the net reproductive rate were higher when individuals were fed on the artificial diet if compared to the host plant, indicating the suitability of the tested diet for the rearing of P. sequax. This artificial diet has been used successfully for the rearing of P. sequax without any fitness effect for over 18 consecutive generations.     

Scalable and flexible inference framework for stochastic dynamic single-cell models

Understanding the inherited nature of how biological processes dynamically change over time and exhibit intra- and inter-individual variability, due to the different responses to environmental stimuli and when interacting with other processes, has been a major focus of systems biology. The rise of single-cell fluorescent microscopy has enabled the study of those phenomena. The analysis of single-cell data with mechanistic models offers an invaluable tool to describe dynamic cellular processes and to rationalise cell-to-cell variability within the population. However, extracting mechanistic information from single-cell data has proven difficult. This requires statistical methods to infer unknown model parameters from dynamic, multi-individual data accounting for heterogeneity caused by both intrinsic (e.g. variations in chemical reactions) and extrinsic (e.g. variability in protein concentrations) noise. Although several inference methods exist, the availability of efficient, general and accessible methods that facilitate modelling of single-cell data, remains lacking. Here we present a scalable and flexible framework for Bayesian inference in state-space mixed-effects single-cell models with stochastic dynamic. Our approach infers model parameters when intrinsic noise is modelled by either exact or approximate stochastic simulators, and when extrinsic noise is modelled by either time-varying, or time-constant parameters that vary between cells. We demonstrate the relevance of our approach by studying how cell-to-cell variation in carbon source utilisation affects heterogeneity in the budding yeast Saccharomyces cerevisiae SNF1 nutrient sensing pathway. We identify hexokinase activity as a source of extrinsic noise and deduce that sugar availability dictates cell-to-cell variability.     

Code Biology – A New Science of Life

Systems Biology and the Modern Synthesis are recent versions of two classical biological paradigms that are known as structuralism and functionalism, or internalism and externalism. According to functionalism (or externalism), living matter is a fundamentally passive entity that owes its organization to external forces (functions that shape organs) or to an external organizing agent (natural selection). Structuralism (or internalism), is the view that living matter is an intrinsically active entity that is capable of organizing itself from within, with purely internal processes that are based on mathematical principles and physical laws. At the molecular level, the basic mechanism of the Modern Synthesis is molecular copying, the process that leads in the short run to heredity and in the long run to natural selection. The basic mechanism of Systems Biology, instead, is self-assembly, the process by which many supramolecular structures are formed by the spontaneous aggregation of their components. In addition to molecular copying and self-assembly, however, molecular biology has uncovered also a third great mechanism at the heart of life. The existence of the genetic code and of many other organic codes in Nature tells us that molecular coding is a biological reality and we need therefore a framework that accounts for it. This framework is Code biology, the study of the codes of life, a new field of research that brings to light an entirely new dimension of the living world and gives us a completely new understanding of the origin and the evolution of life.     

The cell biology of aging

One of the original hypotheses of organismal longevity posits that aging is the natural result of entropy on the cells, tissues, and organs of the animal—a slow, inexorable slide into nonfunctionality caused by stochastic degradation of its parts. We now have evidence that aging is instead at least in part genetically regulated. Many mutations have been discovered to extend lifespan in organisms of all complexities, from yeast to mammals. The study of metazoan model organisms, such as Caenorhabditis elegans, has been instrumental in understanding the role of genetics in the cell biology of aging. Longevity mutants across the spectrum of model organisms demonstrate that rates of aging are regulated through genetic control of cellular processes. The regulation and subsequent breakdown of cellular processes represent a programmatic decision by the cell to either continue or abandon maintenance procedures with age. Our understanding of cell biological processes involved in regulating aging have been particularly informed by longevity mutants and treatments, such as reduced insulin/IGF-1 signaling and dietary restriction, which are critical in determining the distinction between causes of and responses to aging and have revealed a set of downstream targets that participate in a range of cell biological activities. Here we briefly review some of these important cellular processes.     

Physical mapping of the rice genome with BACs

The development of genetics in this century has been catapulted forward by several milestones: rediscovery of Mendel's laws, determination of DNA as the genetic material, discovery of the double-helix structure of DNA and its implications for genetic behavior, and most recently, analysis of restriction fragment length polymorphisms (RFLPs). Each of these milestones has generated a huge wave of progress in genetics. Consequently, our understanding of organismal genetics now extends from phenotypes to their molecular genetic basis. It is now clear that the next wave of progress in genetics will hinge on genome molecular physical mapping, since a genome physical map will provide an invaluable, readily accessible system for many detailed genetic studies and isolation of many genes of economic or biological importance. Recent development of large-DNA fragment (>100 kb) manipulation and cloning technologies, such as pulsed-field gel electrophoresis (PFGE), and yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning, has provided the powerful tools needed to generate molecular physical maps for higher-organism genomes. This chapter will discuss (1) an ideal physical map of plant genome and its applications in plant genetic and biological studies, (2) reviews on physical mapping of the genomes of Caenorhabditis elegans, Arabidopsis thaliana, and man, (3) large-insert DNA libraries: cosmid, YAC and BAC, and genome physical mapping, (4) physical mapping of the rice genome with BACs, and (5) perspectives on the physical mapping of the rice genome with BACs.     

Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology.     

Decontamination of ochratoxin A by yeasts: possible approaches and factors leading to toxin removal in wine

Biological decontamination of mycotoxins using microorganisms is one of the well-known strategies for the management of mycotoxins in foods and feeds. Yeasts are an efficient biosorbant, used in winemaking to reduce the concentration of harmful substances from the must which affect alcoholic fermentation (medium-chain fatty acids) or which affect wine quality in a negative way (ethyl phenols and sulphur products). In recent years, several studies have demonstrated the ability of yeasts to remove ochratoxin A (OTA) by live cells, cell walls and cell wall extracts, yeast lees. In spite of the physical and chemical methods applied to remove the toxin, the biological removal is considered a promising solution, since it is possible to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food. In addition, adsorption is recognized as economically viable, technically feasible and socially acceptable. This paper intends to review the current achievements of OTA removal mediated by yeasts, the recent updates in the selection of strains acting at the same time as starters and as biological tools to remove OTA and the factors affecting the removal process.     

Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl,Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae

Biological synthesis of pharmaceuticals and biochemicals offers an environmentally friendly alternative to conventional chemical synthesis. These alternative methods require the design of metabolic pathways and the identification of enzymes exhibiting adequate activities. Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates are natural metabolites which possess beneficial activities for human health, and the search is expanding for novel derivatives that might have enhanced biological activity. For example, biosynthesis in Dianthus caryophyllus is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT. Here we report that, by exploiting the substrate flexibility of both 4CL5 and HCBT, we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates. Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules. This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases.     

Arabidopsis TRANSCURVATA1 Encodes NUP58, a Component of the Nucleopore Central Channel

The selective trafficking of proteins and RNAs through the nuclear envelope regulates nuclear-cytoplasmic segregation of macromolecules and is mediated by nucleopore complexes (NPCs), which consist of about 400 nucleoporins (Nups) of about 30 types. Extensive studies of nucleoporin function in yeast and vertebrates showed that Nups function in nucleocytoplasmic trafficking and other processes. However, limited studies of plant Nups have identified only a few mutations, which cause pleiotropic phenotypes including reduced growth and early flowering. Here, we describe loss-of-function alleles of Arabidopsis TRANSCURVATA1 (TCU1); these mutations cause increased hypocotyl and petiole length, reticulate and asymmetrically epinastic leaf laminae of reduced size, and early flowering. TCU1 is transcribed in all of the organs and tissues examined, and encodes the putative ortholog of yeast and vertebrate Nup58, a nucleoporin of the Nup62 subcomplex. Nup58 forms the central channel of the NPC and acts directly in translocation of proteins through the nuclear envelope in yeast and vertebrates. Yeast two-hybrid (Y2H) assays identified physical interactions between TCU1/NUP58 and 34 proteins, including nucleoporins, SCF (Skp1/Cul1/F-box) ubiquitin ligase complex components and other nucleoplasm proteins. Genetic interactions were also found between TCU1 and genes encoding nucleoporins, soluble nuclear transport receptors and components of the ubiquitin-proteasome and auxin signaling pathways. These genetic and physical interactions indicate that TCU1/NUP58 is a member of the Nup62 subcomplex of the Arabidopsis NPC. Our findings also suggest regulatory roles for TCU1/NUP58 beyond its function in nucleocytoplasmic trafficking, a hypothesis that is supported by the Y2H and genetic interactions that we observed.     

Diversity of cultivable Azotobacter in the semi-arid alfisol receiving long-term organic and inorganic nutrient amendments

Monitoring the biological processes and microbial diversity is essential for sustaining the soil health for long-term productivity. In the present study, the impact of long-term nutrient management systems on changes in Azotobacter diversity of Indian semi-arid alfisol was assessed. Three soils, i.e., unfertilized control, soils amended with organic manures (OM), and with inorganic chemical fertilizers (IC) from century-old experimental fields were evaluated for Azotobacter diversity by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Bray–Curtis’s similarity index of the ARDRA data of the isolates was analyzed by non-metric multi-dimensional scaling and hierarchical cluster analysis. The results revealed that the long-term organically managed soil recorded significantly higher soil organic carbon, microbial biomass carbon, and total culturable bacterial counts, whereas the chemical fertilized and control soils remained unaffected. Though the Azotobacter population was significantly higher in OM soil than IC and control soils, the genetic diversity was unaffected due to long-term addition of either organic manures or inorganic chemical fertilizers. This result implies the importance of continuous addition of organic manures and also the optimal use of inorganic chemical fertilizers without disturbing the biological properties of the soil.     

Cathodes as electron donors for microbial metabolism: Which extracellular electron transfer mechanisms are involved?

This review illuminates extracellular electron transfer mechanisms that may be involved in microbial bioelectrochemical systems with biocathodes. Microbially-catalyzed cathodes are evolving for new bioprocessing applications for waste(water) treatment, carbon dioxide fixation, chemical product formation, or bioremediation. Extracellular electron transfer processes in biological anodes, were the electrode serves as electron acceptor, have been widely studied. However, for biological cathodes the question remains: what are the biochemical mechanisms for the extracellular electron transfer from a cathode (electron donor) to a microorganism? This question was approached by not only analysing the literature on biocathodes, but also by investigating known extracellular microbial oxidation reactions in environmental processes. Here, it is predicted that in direct electron transfer reactions, c-type cytochromes often together with hydrogenases play a critical role and that, in mediated electron transfer reactions, natural redox mediators, such as PQQ, will be involved in the bioelectrochemical reaction. These mechanisms are very similar to processes at the bioanode, but the components operate at different redox potentials. The biocatalyzed cathode reactions, thereby, are not necessarily energy conserving for the microorganism.     

Biotechnological production of sphingoid bases and their applications

Sphingolipids are not only essential components of biological membranes but also play numerous other vital functions in living cells. Moreover, they are major constituents of the outermost layer of human epidermis which acts as permeability barrier of the skin. Therefore, they have a high potential to be used in a wide variety of application fields such as antibacterial and antifungal agents, active pharmaceutical ingredients of therapeutics as well as active ingredients in cosmeceutical or nutraceutical formulations. However, their chemical synthesis is a complex and cost-intensive process. As the yeast Wickerhamomyces ciferrii has been found to be a natural producer of acetylated sphingoid bases, it provides a promising alternative for their biotechnological synthesis. In the last years, this yeast has been established by classical strain improvements as well as modern genetic engineering for the industrial production of phytosphingosine. Moreover, routes for the synthesis of sphinganine and sphingosine have been implemented. This mini-review summarizes the current knowledge about biosynthesis of sphingoid bases, genetic engineering of W. ciferrii for their biotechnological production, as well as their applications in cosmetic formulations.     

Adenosine Monophosphate Affects Competence Development and Plasmid DNA Transformation in Escherichia coli

Artificial plasmid DNA transformation of Escherichia coli induced by calcium chloride is a routine technique in molecular biology and genetic engineering processes, but its mechanism has remained elusive. Because adenosine monophosphate (AMP) has been found to regulate natural transformation in Haemophilus influenza, we aimed to investigate the effects of AMP and its derivatives on E. coli transformation by treating competence with different concentrations of them. Analysis of the transformation efficiencies revealed that AMP inhibited the artificial plasmid DNA transformation of E. coli in a concentration- and time-dependent manner. Furthermore, we found that AMP had no effect on the expression of the transformed gene but that the intracellular AMP level of the competent cells rose after a 6 h treatment. These results suggested that the intracellular AMP level had an important role in E. coli transformation. And these have useful implications for the further investigation of the mechanism of E. coli transformation.     

TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes^1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons^1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene⁴. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)⁵ (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism⁶ and other representative of the genus Vibrio such as V. fischeri⁷. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region⁸. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences⁵. Several optimization steps concerning the initial protocol of chitin-induced natural transformation⁶ are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes⁸, the donation of PCR-derived DNA as transforming material⁹, and the addition of FLP-recombination target sites (FRT)⁵. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase¹⁰.     

How do biomolecular systems speed up and regulate rates?

The viability of a biological system depends upon careful regulation of the rates of various processes. These rates have limits imposed by intrinsic chemical or physical steps (e.g., diffusion). These limits can be expanded by interactions and dynamics of the biomolecules. For example, (a) a chemical reaction is catalyzed when its transition state is preferentially bound to an enzyme; (b) the folding of a protein molecule is speeded up by specific interactions within the transition-state ensemble and may be assisted by molecular chaperones; (c) the rate of specific binding of a protein molecule to a cellular target can be enhanced by mechanisms such as long-range electrostatic interactions, nonspecific binding and folding upon binding; (d) directional movement of motor proteins is generated by capturing favorable Brownian motion through intermolecular binding energy; and (e) conduction and selectivity of ions through membrane channels are controlled by interactions and the dynamics of channel proteins. Simple physical models are presented here to illustrate these processes and provide a unifying framework for understanding speed attainment and regulation in biomolecular systems.     

Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment

Although genetic engineering techniques for baker's yeast might improve the yeast's fermentation characteristics, the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health prevent their commercial use. Disruption of acid trehalase gene (ATH1) improves freeze tolerance, which is a crucial characteristic in frozen-dough baking. In this study, ATH1 disruptants constructed by genetic modification (GM) and self-cloning (SC) techniques were used as models to study such effects because these strains have higher freeze tolerance and are expected to be used commercially. Behavior of the strains in simulated natural environments, namely, in soil and water, was studied by measuring the change in the number of viable cells and in the concentration of DNA that contains ATH1 loci. Measurements were made using a real-time PCR method during 40 days of cultivation. Results showed that the number of viable cells of GM and SC strains decreased in a time-dependent manner and that the decrease rate was nearly equal to or higher than that for wild-type (WT) yeast. For all three strains (SC, GM, and WT) in the two simulated natural environments (water and soil), the DNA remained longer than did viable cells but the decrease patterns of either the DNA or the viable cells of SC and GM strains had tendencies similar to those of the WT strain. In conclusion, disruption of ATH1 by genetic engineering apparently does not promote the survival of viable cells and DNA in natural environments.     

Cyanophage Diversity, Inferred from g20 Gene Analyses, in the Largest Natural Lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Yeast Assay Highlights the Intrinsic Genomic Instability of Human PML Intron 6 over Intron 3 and the Role of Replication Fork Proteins

Human acute promyelocytic leukemia (APL) is characterized by a specific balanced translocation t(15;17)(q22;q21) involving the PML and RARA genes. In both de novo and therapy-related APL, the most frequent PML breakpoints are located within intron 6, and less frequently in intron 3; the precise mechanisms by which these breakpoints arise and preferentially in PML intron 6 remain unsolved. To investigate the intrinsic properties of the PML intron sequences in vivo, we designed Saccharomyces cerevisiae strains containing human PML intron 6 or intron 3 sequences inserted in yeast chromosome V and measured gross chromosomal rearrangements (GCR). This approach provided evidence that intron 6 had a superior instability over intron 3 due to an intrinsic property of the sequence and identified the 3’ end of intron 6 as the most susceptible to break. Using yeast strains invalidated for genes that control DNA replication, we show that this differential instability depended at least upon Rrm3, a DNA helicase, and Mrc1, the human claspin homolog. GCR induction by hydrogen peroxide, a general genotoxic agent, was also dependent on genetic context. We conclude that: 1) this yeast system provides an alternative approach to study in detail the properties of human sequences in a genetically controlled situation and 2) the different susceptibility to produce DNA breaks in intron 6 versus intron 3 of the human PML gene is likely due to an intrinsic property of the sequence and is under replication fork genetic control.     

An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to Type IV pilus expression

Although natural genetic transformation is a widely disseminated form of genetic exchange in prokaryotic species, the proficiencies with which DNA recognition, uptake and processing occur in nature vary greatly. However, the molecular factors and interactions underlying intra- and interspecies diversity in levels of competence for natural genetic transformation are poorly understood. In Neisseria gonorrhoeae, the Gram-negative aetiologic agent of gonorrhoea, DNA binding and uptake involve components required for Type IV pilus (Tfp) biogenesis as well as those which are structurally related to Tfp biogenesis components but dispensable for organelle expression. We demonstrate here that the gonococcal PilV protein, structurally related to Tfp pilin subunits, is an intrinsic inhibitor of natural genetic transformation which acts ultimately by reducing the levels of sequence-specific DNA uptake into the cell. Specifically, we show that DNA uptake is enhanced in strains bearing pilV mutations and reduced in strains overexpressing PilV. Furthermore, we show that PilV exerts its effect by acting as an antagonist of ComP, a positive effector of sequence-specific DNA binding. As it prevents the accumulation of ComP at a site where it can be purified by shear extraction of intact cells, the data are most consistent with PilV either obstructing ComP trafficking or altering ComP stability. In addition, we report that ComP and PilV play overlapping and partially redundant roles in Tfp biogenesis and document other genetic interactions between comP and pilV together with the pilE and pilT genes required for the expression of retractile Tfp. Together, the results reveal a novel mechanism by which the levels of competence are governed in prokaryotic species and suggest unique ways by which competence might be modulated.