Peptide design by artificial neural networks and computer-based evolutionary search

A technique for systematic peptide variation by a combination of rational and evolutionary approaches is presented. The design scheme consists of five consecutive steps: (i) identification of a “seed peptide” with a desired activity, (ii) generation of variants selected from a physicochemical space around the seed peptide, (iii) synthesis and testing of this biased library, (iv) modeling of a quantitative sequence-activity relationship by an artificial neural network, and (v) de novo design by a computer-based evolutionary search in sequence space using the trained neural network as the fitness function. This strategy was successfully applied to the identification of novel peptides that fully prevent the positive chronotropic effect of anti-β1-adrenoreceptor autoantibodies from the serum of patients with dilated cardiomyopathy. The seed peptide, comprising 10 residues, was derived by epitope mapping from an extracellular loop of human β1-adrenoreceptor. A set of 90 peptides was synthesized and tested to provide training data for neural network development. De novo design revealed peptides with desired activities that do not match the seed peptide sequence. These results demonstrate that computer-based evolutionary searches can generate novel peptides with substantial biological activity.

Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [D6S257, logarithm of odds (lod) = 3.90, P = 0.000011] and at three additional regions: 16q13 (D16S415, lod = 3.64, P = 0.000022), 14q21–23 (D14S276, lod = 2.81, P = 0.00016), and 20p12 (D20S186, lod = 2.62, P = 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21–33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores ≥1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum genehunter-plus nonparametric linkage score = 4.05, P = 5.22 × 10−4; sibpal Pempirical value <3 × 10−5) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum genehunter-plus nonparametric linkage score = 3.29, P = 2.57 × 10−3; sibpal Pempirical value <1 × 10−3) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.

Topological testing of the mechanism of homology search promoted by RecA protein

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.

Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

Computational method to reduce the search space for directed protein evolution

We introduce a computational method to optimize the in vitro evolution of proteins. Simulating evolution with a simple model that statistically describes the fitness landscape, we find that beneficial mutations tend to occur at amino acid positions that are tolerant to substitutions, in the limit of small libraries and low mutation rates. We transform this observation into a design strategy by applying mean-field theory to a structure-based computational model to calculate each residue's structural tolerance. Thermostabilizing and activity-increasing mutations accumulated during the experimental directed evolution of subtilisin E and T4 lysozyme are strongly directed to sites identified by using this computational approach. This method can be used to predict positions where mutations are likely to lead to improvement of specific protein properties.

Visual constraints in foraging bumblebees: Flower size and color affect search time and flight behavior

In optimal foraging theory, search time is a key variable defining the value of a prey type. But the sensory-perceptual processes that constrain the search for food have rarely been considered. Here we evaluate the flight behavior of bumblebees (Bombus terrestris) searching for artificial flowers of various sizes and colors. When flowers were large, search times correlated well with the color contrast of the targets with their green foliage-type background, as predicted by a model of color opponent coding using inputs from the bees' UV, blue, and green receptors. Targets that made poor color contrast with their backdrop, such as white, UV-reflecting ones, or red flowers, took longest to detect, even though brightness contrast with the background was pronounced. When searching for small targets, bees changed their strategy in several ways. They flew significantly slower and closer to the ground, so increasing the minimum detectable area subtended by an object on the ground. In addition, they used a different neuronal channel for flower detection. Instead of color contrast, they used only the green receptor signal for detection. We relate these findings to temporal and spatial limitations of different neuronal channels involved in stimulus detection and recognition. Thus, foraging speed may not be limited only by factors such as prey density, flight energetics, and scramble competition. Our results show that understanding the behavioral ecology of foraging can substantially gain from knowledge about mechanisms of visual information processing.

Target gene search for the metal-responsive transcription factor MTF-1

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding α-fetoprotein, the liver-enriched transcription factor C/EBPα and tear lipocalin/von Ebner’s gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns γ-glutamylcysteine synthetasehc (γ-GCShc), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. γ-GCShc mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

In search of planets and life around other stars

The discovery of over a dozen low-mass companions to nearby stars has intensified scientific and public interest in a longer term search for habitable planets like our own. However, the nature of the detected companions, and in particular whether they resemble Jupiter in properties and origin, remains undetermined.