Improvement of yield of Pleurotus eryngii var. eryngii by substrate supplementation and use of a casing overlay

Improved yield and biological efficiency (BE) of Pleurotus eryngii var. eryngii were achieved by supplementation of substrate with a commercial delayed-release nutrient and use of a casing overlay. Yield increases of 14% were achieved from cased substrates that were supplemented at time of casing with delayed-release nutrient (Remo’s). Use of a casing layer enhanced yield by 141% over non-cased substrates. When casing and substrate supplementation were combined, yield increased 179% over non-cased/non-supplemented substrates. Mushrooms harvested from cased substrates were darker in color and solids contents were lower compared to non-cased substrates. An additional break of mushrooms was harvested from non-cased “spent” substrate by fragmenting and re-supplementing the substrate prior to the application of a casing overlay. Three production methods were compared for their effect on mushroom yield: “standard”, “casing” and “casing after first break”. Casing of the substrate before first break (“casing” production method) resulted in the highest yield and biological efficiency.     

Are Victoria West cores “proto-Levallois”? A phylogenetic assessment

Cores from South Africa assigned to the “Victoria West” industry have long been purported as a “proto-Levallois” core form, and thus regarded as ancestral to the Levallois prepared core technologies of the Middle Paleolithic and African Middle Stone Age. Similarities in form between Victoria West cores, in terms of surface morphology and the removal of large flakes from a prepared surface, led to hypothesized schemes of technological evolution from Victoria West cores through to fully developed Levallois cores. However, the phylogenetic basis of this Victoria West “proto-Levallois” hypothesis, and the assumptions of phylogenetic homology upon which it rests, have never been tested formally. In recent years, archaeologists have begun to use phylogenetic methods drawn from biology to test hypotheses of technological and cultural evolution. Here, the phylogenetic assumptions of the Victoria West “proto-Levallois” hypothesis are tested directly using a cladistic (maximum parsimony) protocol. The cladistic analyses indicate that Victoria West cores are not the basal sister taxon of a Levallois clade, as predicted by the proto-Levallois hypothesis. Moreover, character analyses demonstrate that several characters relating to core surfaces and flake scar morphology are not phylogenetically homologous, but result from convergent technological evolution within the Acheulean techno-complex. Post hoc analyses further determine that these results are not confounded by choice of outgroup or raw material factors. The results were also shown to be robust on the basis of the ensemble retention index statistic, bootstrap analyses, and permutation tests. Hence, it is concluded that Victoria West cores do not represent a “proto-Levallois” core form, and that the term “para-Levallois” should more correctly be applied on phylogenetic grounds. It is further argued that even in cases where different technologies are found to share phylogenetically homologous features, use of the term “proto” is questionable on theoretical grounds.     

Similarity selection and the evolution of sex: revisiting the red queen

For over 25 years, many evolutionary ecologists have believed that sexual reproduction occurs because it allows hosts to change genotypes each generation and thereby evade their coevolving parasites. However, recent influential theoretical analyses suggest that, though parasites can select for sex under some conditions, they often select against it. These models assume that encounters between hosts and parasites are completely random. Because of this assumption, the fitness of a host depends only on its own genotype (“genotypic selection”). If a host is even slightly more likely to encounter a parasite transmitted by its mother than expected by random chance, then the fitness of a host also depends on its genetic similarity to its mother (“similarity selection”). A population genetic model is presented here that includes both genotypic and similarity selection, allowing them to be directly compared in the same framework. It is shown that similarity selection is a much more potent force with respect to the evolution of sex than is genotypic selection. Consequently, similarity selection can drive the evolution of sex even if it is much weaker than genotypic selection with respect to fitness. Examination of explicit coevolutionary models reveals that even a small degree of mother–offspring parasite transmission can cause parasites to favor sex rather than oppose it. In contrast to previous predictions, the model shows that weakly virulent parasites are more likely to favor sex than are highly virulent ones. Parasites have figured prominently in discussions of the evolution of sex, but recent models suggest that parasites often select against sex rather than for it. With the inclusion of small and realistic exposure biases, parasites are much more likely to favor sex. Though parasites alone may not provide a complete explanation for sex, the results presented here expand the potential for parasites to contribute to the maintenance of sex rather than act against it.     

Emergence of novel enzyme quasi-species depends on the substrate matrix

Current research on enzyme evolution has shown that many enzymes are promiscuous and have activities with alternative substrates. Mutagenesis tends to relax substrate selectivity, and evolving enzymes can be regarded (summed over evolutionary time) as clusters of enzyme variants, or “quasi-species,” tested against a “substrate matrix” defined by all chemical substances to which the evolvants are exposed.In this investigation, the importance of the substrate matrix for identification of evolvable clusters of enzymes was evaluated by random sampling of variants from a library of glutathione transferase (GST) mutants. The variant GSTs were created by DNA shuffling of homologous Alpha class sequences. The substrate matrix was an array of alternative substrates used under defined experimental conditions. The measured enzyme activities produced a rectangular matrix, in which the rows can be projected as enzyme vectors in substrate-activity space and, reciprocally, the columns can be projected as alternative substrate vectors in enzyme-activity space. Multivariate analysis of the catalytic activities demonstrated that the enzyme vectors formed two primary clusters or functional “molecular quasi-species.” These quasi-species serve as the raw material from which more specialized enzymes eventually could evolve. The substrate vectors similarly formed two major groups. Identification of separate quasi-species of GSTs in a mutant library was critically dependent on the nature of the substrate matrix. When substrates from just one of the two groups were used, only one cluster of enzymes could be recognized. On the other hand, expansion of the substrate matrix to include additional substrates showed the presence of a third quasi-species among the GST variants already analyzed. Thus, the portrayal of the functional quasi-species is intimately linked to the effective substrate matrix. In natural evolution, the substrates actually encountered therefore play a pivotal role in determining whether latent catalytic abilities become manifest in novel enzymes.     

Lipid outward translocation by ABC proteins

In humans, about 50 ABC proteins play physiologically important roles. Many ABC proteins are involved in lipid outward translocation and lipid homeostasis in the body, and defects in their functions cause various diseases. However, the precise mechanisms of substrate transport remain unclear. In bacteria, several ABC proteins are involved in the transport of lipoproteins and lipopolysaccharides from the inner to outer membrane, and their functioning is a prerequisite for survival. Their functions can be divided into “flip-flop” and “projection”. In this review, human ABC proteins are compared to bacterial proteins to elucidate their mechanisms.     

Linkage rules for plant-pollinator networks: trait complementarity or exploitation barriers?

Recent attempts to examine the biological processes responsible for the general characteristics of mutualistic networks focus on two types of explanations: nonmatching biological attributes of species that prevent the occurrence of certain interactions (“forbidden links”), arising from trait complementarity in mutualist networks (as compared to barriers to exploitation in antagonistic ones), and random interactions among individuals that are proportional to their abundances in the observed community (“neutrality hypothesis”). We explored the consequences that simple linkage rules based on the first two hypotheses (complementarity of traits versus barriers to exploitation) had on the topology of plant–pollination networks. Independent of the linkage rules used, the inclusion of a small set of traits (two to four) sufficed to account for the complex topological patterns observed in real-world networks. Optimal performance was achieved by a “mixed model” that combined rules that link plants and pollinators whose trait ranges overlap (“complementarity models”) and rules that link pollinators to flowers whose traits are below a pollinator-specific barrier value (“barrier models”). Deterrence of floral parasites (barrier model) is therefore at least as important as increasing pollination efficiency (complementarity model) in the evolutionary shaping of plant–pollinator networks.     

Beta-specificity: The turnover of host species in space and another way to measure host specificity

Host specificity is often measured as the number of host species used by a parasite, or as their phylogenetic diversity; both of these measures ignore the larger scale component of host use by parasites. A parasite may exploit very few host species in one locality but these hosts may be substituted for completely different species elsewhere; in contrast, another parasite may exploit many host species in one locality, with the identity of these hosts remaining the same throughout the parasite’s geographical range. To capture these spatial nuances of host specificity, we propose to use an index for host species turnover across localities, or beta-specificity (β_SPF), that is derived from studies of spatial patterns in plant and animal diversity. We apply this index to fleas parasitic on small mammals to show that: (i) it is statistically independent of traditional or “local” measures of host specificity as well as of “global” measures of host specificity, and (ii) it is also independent of the size of the geographical area studied or the sampling effort put into collecting hosts and parasites. Furthermore, the distribution of β_SPF values among flea species shows a significant phylogenetic signal, i.e. related flea species have more similar β_SPF values than expected by chance. Nevertheless, most possible combinations of either local specificity (alpha-specificity) or global (gamma-specificity) and beta-specificity are observed among flea species, suggesting that adding a spatial component to studies of host use reveals a new facet of specificity. The measure presented here provides a new perspective on host specificity on a scale relevant to studies on topics ranging from biogeography to evolution and may underlie the rate and extent of disease transmission and population dynamics.     

An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the “gatekeeper” residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3^M385G can also specifically utilize N⁶-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.     

Codon sextets with leading role of serine create “ideal” symmetry classification scheme of the genetic code

The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator.     

Ampelozyziphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria in the Amazon Region, hampers the development of Plasmodium berghei sporozoites

Most medicinal plants used against malaria in endemic areas aim to treat the acute symptoms of the disease such as high temperature fevers with periodicity and chills. In some endemic areas of the Brazilian Amazon region one medicinal plant seems to be an exception: Ampelozyziphus amazonicus, locally named “Indian beer” or “Saracura-mira”, used to prevent the disease when taken daily as a cold suspension of powdered dried roots. In previous work we found no activity of the plant extracts against malaria blood parasites in experimentally infected animals (mice and chickens) or in cultures of Plasmodium falciparum. However, in infections induced by sporozoites, chickens treated with plant extracts were partially protected against Plasmodium gallinaceum and showed reduced numbers of exoerythrocytic forms in the brain. We now present stronger evidence that the ethanolic extract of “Indian beer” roots hampers in vitro and in vivo development of Plasmodium berghei sporozoites, a rodent malaria parasite. Some mice treated with high doses of the plant extract did not become infected after sporozoite inoculation, whereas others had a delayed prepatent period and lower parasitemia. Our data validates the use of “Indian beer” as a remedy for malaria prophylaxis in the Amazon, where the plant exists and the disease represents an important problem which is difficult to control. Studies aiming to identify the active compounds responsible for the herein described causal prophylactic activity are needed and may lead to a new antimalarial prophylactic.     

A structure–activity study to identify novel and efficient substrates of the human semicarbazide-sensitive amine oxidase/VAP-1 enzyme

Kinetic studies were performed with various alkanamines as “substrate probes” of the properties of the active site of the human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). We found that the enzyme–substrate recognition step is mainly controlled by apolar interactions and that a “good” substrate has a molecular structure containing a long aliphatic chain and a second positive charge at a distance greater than 12 ? from the reactive amino group. In this context, we identified a novel substrate for the human SSAO/VAP-1, 1,12-diaminododecane (DIADO), which is characterised by the highest catalytic efficiency reported to date in comparison to the prototypic substrate benzylamine. Computational docking studies revealed the structural basis of this behaviour, highlighting the key role played by Lys393 in hindering substrate docking.     

The crystal structure analysis of group B Streptococcus sortase C1: a model for the "lid" movement upon substrate binding

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible “lid” anchored in the active site. However, the mechanistic details of the “lid” displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its “lid” mutant and proposed a role of the “lid” as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.     

Coexistence of individual and social learners during range expansion

Individual learning and social learning are two primary abilities supporting cultural evolution. Conditions for their evolution have mostly been studied by investigating gene frequency dynamics, which essentially implies constant population size. Predictions from such “static” models may only be of partial relevance to the evolution of advanced individual learning in modern humans, because modern humans have experienced rapid population growth and range expansion during “out-of-Africa.” Here we model the spatial population dynamics of individual and social learners by a reaction–diffusion system. One feature of our model is the inclusion of the possibility that social learners may fail to find an exemplar to copy in regions where the population density is low. Due to this attenuation effect, the invasion speed of social learners is diminished, and various kinds of invasion dynamics are observed. Our primary findings are: (1) individual learners can persist indefinitely when invading environmentally homogeneous infinite space; (2) the occurrence of individual learners at the front may inhibit the spread of social learners. These results suggest that “out-of-Africa” may have driven the evolution of advanced individual learning ability in modern humans.     

Structural and chemical profiling of the human cytosolic sulfotransferases

The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

L'analogie et la représentation de l'art primitif à la fin du XIX siècle

In this paper, we will examine the foundations of Western representation of Paleolithic art at the end of the nineteenth century. Taking the period of 1864-1902 into account, we will prove the leading role of analogy between “modern primitive societies” and “prehistoric societies” in the very definition of “primitive art”. According to us, the representation of the “primitive artist” at that time was largely based in comparison between art which came from modern primitive societies living in Africa, Australia or America, and prehistoric art which was authenticated at about 1865. Through this examination, we will show the way in which analogy functions as a main category in the construction of scientific knowledge.     

A straightforward kinetic evidence for coexistence of “induced fit” and “selected fit” in the reaction mechanism of a mutant tryptophan indole lyase Y72F from Proteus vulgaris

The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-l-alanine (OIA), with the natural substrate, l-tryptophan, and with a substrate S-ethyl-l-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme (“induced fit”) is realized in the case of mutant Y72F enzyme reaction with OIA. For the reaction of mutant enzyme with l-Trp at lower concentrations of the latter a principally different kinetic scheme is observed. This scheme suggests that binding of the substrate and formation of the quinonoid intermediate are at fast equilibrium, while preceding conformational changes of the holoenzyme proceed with measureable rates (“selected fit”). For the reaction with S-ethyl-l-cysteine the observed concentration dependence of k_obs agrees with the realization of both kinetic schemes, the “selected fit” becoming predominant at lower concentrations of substrate, the “induced fit”— at higher ones. In the reaction with S-ethyl-l-cysteine the formation of the quinonoid intermediate proceeds slower than does catalytic α,β-elimination of ethylthiol from S-ethyl-l-cysteine, and consequently does not play a considerable role in the catalysis, which may be effected by a concerted E2 mechanism.     

The quest for molecular quasi-species in ligand-activity space and its application to directed enzyme evolution

We propose that the proper evolving unit in enzyme evolution is not a single “fittest molecule”, but a cluster of related variants denoted a “quasi-species”. A distribution of variants provides genetic variability and thereby reduces the risk of inbreeding and evolutionary dead-ends. Different matrices of substrates or activity modulators will lead to different selection criteria and divergent evolutionary trajectories. We provide examples from our directed evolution of glutathione transferases illustrating the interplay between libraries of enzyme variants and ligand matrices in the identification of quasi-species. The ligand matrix is shown to be crucial to the outcome of the search for novel activities. In this sense the experimental system resembles the biological environment in governing the evolution of enzymes.