Matched pairs in factorial experiments with binary data

Two more models are proposed for the analysis of matched pairs in factorial experiments with binary data. They are applied to two-period crossover designs. Estimation and test procedures are derived both approximately and by maximizing likelihoods. Comparisons are made with previous methods of analysis and between the models proposed.     

Two models of the recognition and detection of texture-defined letters compared

We quantified texture segregation by measuring psychophysically the percentage correct detection scores for each of a set of 10 texture-defined (TD) letters using the temporal two-alternative forced choice method, and at the same time quantified spatial discrimination of the TD form of measuring psychophysically the percentage correct letter recognition scores for the 10 letters. Ten levels of task difficulty were created by adding noise dots to the texture patterns. The resulting psychophysical data were used to test and compare models of the detection and recognition of texture-defined letters. Each model comprised a sequence of physiologically plausible stages in early visual processing. Each had the same first, second and third stages, namely linear orientation-tuned spatial filters followed by rectification and smoothing. Model 1 had only one non-linear stage. Model 2 had two non-linear stages. In model 2 the second non-linear stage was cross-orientation inhibition. This second non-linear stage enhanced the texture borders by, in effect, comparing textures at different locations in the texture pattern. In both models, the last stage modelled either letter detection or letter recognition. Letter recognition was modelled as follows. We passed a given letter stimulus through the first several stages of a model and, in 10 separate calculations, cross-correlated the output with a template of each of the 10 letters. From these 10 correlations we obtained a predicted percentage correct letter recognition score for the given letter stimulus. The predicted recognition scores closely agreed with the experimental data at all 10 levels of task difficulty for model 2, but not for model 1. We conclude that a borderenhancing algorithm is necessary to model letter recognition. The letter-detection algorithm modelled detection of part of a letter (a single letter stroke) in terms of the signal-to-noise ratio of a letter-segment detector. The predicted letter detection scores fitted the data closely for both models.     

Activation and molecular recognition of the GPCR rhodopsin - insights from time-resolved fluorescence depolarisation and single molecule experiments

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.     

Bayesian action-perception computational model: interaction of production and recognition of cursive letters

In this paper, we study the collaboration of perception and action representations involved in cursive letter recognition and production. We propose a mathematical formulation for the whole perception-action loop, based on probabilistic modeling and bayesian inference, which we call the Bayesian Action-Perception (BAP) model. Being a model of both perception and action processes, the purpose of this model is to study the interaction of these processes. More precisely, the model includes a feedback loop from motor production, which implements an internal simulation of movement. Motor knowledge can therefore be involved during perception tasks. In this paper, we formally define the BAP model and show how it solves the following six varied cognitive tasks using bayesian inference: i) letter recognition (purely sensory), ii) writer recognition, iii) letter production (with different effectors), iv) copying of trajectories, v) copying of letters, and vi) letter recognition (with internal simulation of movements). We present computer simulations of each of these cognitive tasks, and discuss experimental predictions and theoretical developments.     

Unstructured intermediate states in single protein force experiments

Recent single-molecule force measurements on single-domain proteins have highlighted a three-state folding mechanism where a stabilized intermediate state (I) is observed on the folding trajectory between the stretched state and the native state. Here we investigate on-lattice protein-like heteropolymer models that lead to a three-state mechanism and show that force experiments can be useful to determine the structure of I. We have mostly found that I is composed of a core stabilized by a high number of native contacts, plus an unstructured extended chain. The lifetime of I is shown to be sensitive to modifications of the protein that spoil the core. We then propose three types of modifications--point mutations, cuts, and circular permutations--aiming at: (1) confirming the presence of the core and (2) determining its location, within one amino acid accuracy, along the polypeptide chain. We also propose force jump protocols aiming to probe the on/off-pathway nature of I.     

Mechanism of homology recognition in DNA recombination from dual-molecule experiments

In E. coli homologous recombination, a filament of RecA protein formed on DNA searches and pairs a homologous sequence within a second DNA molecule with remarkable speed and fidelity. Here, we directly probe the strength of the two-molecule interactions involved in homology search and recognition using dual-molecule manipulation, combining magnetic and optical tweezers. We find that the filament's secondary DNA-binding site interacts with a single strand of the incoming double-stranded DNA during homology sampling. Recognition requires opening of the helix and is strongly promoted by unwinding torsional stress. Recognition is achieved upon binding of both strands of the incoming dsDNA to each of two ssDNA-binding sites in the filament. The data indicate a physical picture for homology recognition in which the fidelity of the search process is governed by the distance between the DNA-binding sites.     

The impact of recombination on short-term selection gain in plant breeding experiments

Recombination is a requirement for response to selection, but researchers still debate whether increasing recombination beyond normal levels will result in significant gains in short-term selection. We tested this hypothesis, in the context of plant breeding, through a series of simulation experiments comparing short-term selection response (≤20 cycles) between populations with normal levels of recombination and similar populations with unconstrained recombination (i.e., free recombination). We considered additive and epistatic models and examined a wide range of values for key design variables: selection cycles, QTL number, heritability, linkage phase, selection intensity and population size. With few exceptions, going from normal to unconstrained levels of recombination produced only modest gains in response to selection (≈11 % on average). We then asked how breeders might capture some of this theoretical gain by increasing recombination through either (1) extra rounds of mating or (2) selection of highly recombinant individuals via use of molecular markers/maps. All methods tested captured less than half of the potential gain, but our analysis indicates that the most effective method is to select for increased recombination and the trait simultaneously. This recommendation is based on evidence of a favorable interaction between trait selection and the impact of recombination on selection gains. Finally, we examined the relative contributions of the two components of meiotic recombination, chromosome assortment and crossing over, to short-term selection gain. Depending primarily on the presence of trait selection pressure, chromosome assortment alone accounted for 40–75 % of gain in response to short-term selection.     

Heterology tolerance and recognition of mismatched base pairs by human Rad51 protein

Human Rad51 (hRad51) promoted homology recognition and subsequent strand exchange are the key steps in human homologous recombination mediated repair of DNA double-strand breaks. However, it is still not clear how hRad51 deals with sequence heterology between the two homologous chromosomes in eukaryotic cells, which would lead to mismatched base pairs after strand exchange. Excessive tolerance of sequence heterology may compromise the fidelity of repair of DNA double-strand breaks. In this study, fluorescence resonance energy transfer (FRET) was used to monitor the heterology tolerance of human Rad51 mediated strand exchange reactions, in real time, by introducing either G-T or I-C mismatched base pairs between the two homologous DNA strands. The strand exchange reactions were much more sensitive to G-T than to I-C base pairs. These results imply that the recognition of homology and the tolerance of heterology by hRad51 may depend on the local structural motif adopted by the base pairs participating in strand exchange. AnhRad51 mutant protein (hRad51K133R), deficient in ATP hydrolysis, showed greater heterology tolerance to both types of mismatch base pairing, suggesting that ATPase activity may be important for maintenance of high fidelity homologous recombination DNA repair.     

HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA

We have used an iterative in vitro genetic selection to identify the important structural features of the viral RNA element bound by the Rev protein of human immunodeficiency virus type 1 (HIV-1). Functional Rev-binding RNAs were selected from a pool of 10(13) variants of the wild-type Rev-binding domain. Bases conserved among the binding species define a 20 nucleotide core binding element. Covariation of some of these conserved bases indicates that the Rev-binding element is a stem-bulge-stem with a G:G base pair in the bulge. Mutational studies show that this non-Watson-Crick base pair is required for Rev binding in vitro and Rev responsiveness in vivo. We propose that the G:G base pair distorts the sugar-phosphate backbone of viral RNA and that this distortion is a critical determinant of recognition by Rev.     

Substituent effects on the pairing and polymerase recognition of simple unnatural base pairs

As part of an effort to develop stable and replicable unnatural base pairs, we have evaluated a large number of unnatural nucleotides with predominantly hydrophobic nucleobases. Despite its limited aromatic surface area, a nucleobase analog scaffold that has emerged as being especially promising is the simple phenyl ring. Modifications of this scaffold with methyl and fluoro groups have been shown to impact base pair stability and polymerase recognition, suggesting that nucleobase shape, hydrophobicity and electrostatics are important. To further explore the impact of heteroatom substitution within this nucleobase scaffold, we report the synthesis, stability and polymerase recognition of nucleoside analogs bearing single bromo- or cyano-derivatized phenyl rings. Both modifications are found to generally stabilize base pair formation to a greater extent than methyl or fluoro substitution. Moreover, polymerase recognition of the unnatural base pairs is found to be very sensitive to both the position and nature of the heteroatom substituent. The results help identify the determinants of base pair stability and efficient replication and should contribute to the effort to develop stable and replicable unnatural base pairs.     

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.