Precursors to natural grammar learning: preliminary evidence from 4-month-old infants

When learning a new language, grammar--although difficult--is very important, as grammatical rules determine the relations between the words in a sentence. There is evidence that very young infants can detect rules determining the relation between neighbouring syllables in short syllable sequences. A critical feature of all natural languages, however, is that many grammatical rules concern the dependency relation between non-neighbouring words or elements in a sentence i.e. between an auxiliary and verb inflection as in is singing. Thus, the issue of when and how children begin to recognize such non-adjacent dependencies is fundamental to our understanding of language acquisition. Here, we use brain potential measures to demonstrate that the ability to recognize dependencies between non-adjacent elements in a novel natural language is observable by the age of 4 months. Brain responses indicate that 4-month-old German infants discriminate between grammatical and ungrammatical dependencies in auditorily presented Italian sentences after only brief exposure to correct sentences of the same type. As the grammatical dependencies are realized by phonologically distinct syllables the present data most likely reflect phonologically based implicit learning mechanisms which can serve as a precursor to later grammar learning.     

Action at a distance: dependency sensitivity in a New World primate

Sensitivity to dependencies (correspondences between distant items) in sensory stimuli plays a crucial role in human music and language. Here, we show that squirrel monkeys (Saimiri sciureus) can detect abstract, non-adjacent dependencies in auditory stimuli. Monkeys discriminated between tone sequences containing a dependency and those lacking it, and generalized to previously unheard pitch classes and novel dependency distances. This constitutes the first pattern learning study where artificial stimuli were designed with the species'' communication system in mind. These results suggest that the ability to recognize dependencies represents a capability that had already evolved in humans’ last common ancestor with squirrel monkeys, and perhaps before.     

A rostro-caudal gradient of structured sequence processing in the left inferior frontal gyrus

In this paper, we present two novel perspectives on the function of the left inferior frontal gyrus (LIFG). First, a structured sequence processing perspective facilitates the search for functional segregation within the LIFG and provides a way to express common aspects across cognitive domains including language, music and action. Converging evidence from functional magnetic resonance imaging and transcranial magnetic stimulation studies suggests that the LIFG is engaged in sequential processing in artificial grammar learning, independently of particular stimulus features of the elements (whether letters, syllables or shapes are used to build up sequences). The LIFG has been repeatedly linked to processing of artificial grammars across all different grammars tested, whether they include non-adjacent dependencies or mere adjacent dependencies. Second, we apply the sequence processing perspective to understand how the functional segregation of semantics, syntax and phonology in the LIFG can be integrated in the general organization of the lateral prefrontal cortex (PFC). Recently, it was proposed that the functional organization of the lateral PFC follows a rostro-caudal gradient, such that more abstract processing in cognitive control is subserved by more rostral regions of the lateral PFC. We explore the literature from the viewpoint that functional segregation within the LIFG can be embedded in a general rostro-caudal abstraction gradient in the lateral PFC. If the lateral PFC follows a rostro-caudal abstraction gradient, then this predicts that the LIFG follows the same principles, but this prediction has not yet been tested or explored in the LIFG literature. Integration might provide further insights into the functional architecture of the LIFG and the lateral PFC.     

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins,PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.     

Does Formal Complexity Reflect Cognitive Complexity? Investigating Aspects of the Chomsky Hierarchy in an Artificial Language Learning Study

This study investigated whether formal complexity, as described by the Chomsky Hierarchy, corresponds to cognitive complexity during language learning. According to the Chomsky Hierarchy, nested dependencies (context-free) are less complex than cross-serial dependencies (mildly context-sensitive). In two artificial grammar learning (AGL) experiments participants were presented with a language containing either nested or cross-serial dependencies. A learning effect for both types of dependencies could be observed, but no difference between dependency types emerged. These behavioral findings do not seem to reflect complexity differences as described in the Chomsky Hierarchy. This study extends previous findings in demonstrating learning effects for nested and cross-serial dependencies with more natural stimulus materials in a classical AGL paradigm after only one hour of exposure. The current findings can be taken as a starting point for further exploring the degree to which the Chomsky Hierarchy reflects cognitive processes.     

A stochastic context free grammar based framework for analysis of protein sequences

Background  

In the last decade, there have been many applications of formal language theory in bioinformatics such as RNA structure prediction and detection of patterns in DNA. However, in the field of proteomics, the size of the protein alphabet and the complexity of relationship between amino acids have mainly limited the application of formal language theory to the production of grammars whose expressive power is not higher than stochastic regular grammars. However, these grammars, like other state of the art methods, cannot cover any higher-order dependencies such as nested and crossing relationships that are common in proteins. In order to overcome some of these limitations, we propose a Stochastic Context Free Grammar based framework for the analysis of protein sequences where grammars are induced using a genetic algorithm.     

Barbieri’s Organic Codes Enable Error Correction of Genomes

Barbieri introduced and developed the concept of organic codes. The most basic of them is the genetic code, a set of correspondence rules between otherwise unrelated sequences: strings of nucleotides on the one hand, polypeptidic chains on the other hand. Barbieri noticed that it implies ‘coding by convention’ as arbitrary as the semantic relations a language establishes between words and outer objects. Moreover, the major transitions in life evolution originated in new organic codes similarly involving conventional rules. Independently, dealing with heredity as communication over time and relying on information theory, we asserted that the conservation of genomes over the ages demands that error-correcting codes make them resilient to casual errors. Moreover, the better conservation of very old parts of the genome demands that they result from combining successively established nested codes such that the older an information, the more numerous component codes protect it. Barbieri’s concept of organic code and that of genomic error-correcting code may seem unrelated. We show however that organic codes actually entail error-correcting properties. Error-correcting, in general, results from constraints being imposed on a set of sequences. Mathematical equalities are conveniently used in communication engineering for expressing constraints but error correction only needs that constraints exist. Biological sequences are similarly endowed with error-correcting ability by physical-chemical or linguistic constraints, thus defining ‘soft codes’. These constraints are moreover presumably efficient for correcting errors. Insofar as biological sequences are subjected to constraints, organic codes necessarily involve soft codes, and their successive onset results in the nested structure we hypothesized. Organic codes are generated and maintained by means of molecular ‘semantic feedback loops’. Each of these loops involves genes which code for proteins, the enzymatic action of which controls a function needed for the protein assembly. Taken together, thus, they control the assembly of their own structure as instructed by the genome and, once closed, these loops ensure their own conservation. However, the semantic feedback loops do not prevent the genome lengthening. It increases both the redundancy of the genome (as an error-correcting code) and the information quantity it bears, thus improving the genome reliability and the specificity of the enzymes, which enables further evolution.     

Splice site prediction with quadratic discriminant analysis using diversity measure

Based on the conservation of nucleotides at splicing sites and the features of base composition and base correlation around these sites we use the method of increment of diversity combined with quadratic discriminant analysis (IDQD) to study the dependence structure of splicing sites and predict the exons/introns and their boundaries for four model genomes: Caenorhabditis elegans, Arabidopsis thaliana, Drosophila melanogaster and human. The comparison of compositional features between two sequences and the comparison of base dependencies at adjacent or non-adjacent positions of two sequences can be integrated automatically in the increment of diversity (ID). Eight feature variables around a potential splice site are defined in terms of ID. They are integrated in a single formal framework given by IDQD. In our calculations 7 (8) base region around the donor (acceptor) sites have been considered in studying the conservation of nucleotides and sequences of 48 bp on either side of splice sites have been used in studying the compositional and base-correlating features. The windows are enlarged to 16 (donor), 29 (acceptor) and 80 bp (either side) to improve the prediction for human splice sites. The prediction capability of the present method is comparable with the leading splice site detector—GeneSplicer.     

Using estimative reaction free energy to predict splice sites and their flanking competitors

A crucial part in the gene structure prediction is to identify the accurate splice sites, not only constitutive but also alternative ones. Here, we use the maximum information principle (MIP) to analyze the conservative segments around splice sites. According to the MIP, a reaction free energy (RFE) expression is deduced, which can be employed to estimate the free energy change during splicing reaction involving a donor or acceptor site. The expression contains not only the background probability factors, but also all kinds of dependencies among both adjacent and non-adjacent bases. We apply the RFE expression to recognize splice sites and their flanking competitors in human genes, the results show high sensitivity and specificity, so the RFE expression accords well with the splicing reaction process. Moreover, the RFE expression is better than previous methods for predicting competitors of splice sites, and it outperforms the reaction free energy subtraction (RFES), that implies RFE competition between a given splice site and its flanking competitor may not be an only primary factor for alternative splice site selection. The work is helpful to not only the understanding of splicing reaction from its relation to MIP, but also the research on computational recognition of splicing sites and alternative splice events.     

Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.

Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3- to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 10(2) or 10(3) proviral molecules and analyzing the product by high-resolution electrophoresis, the total number and abundance of distinct length variants within an individual could be estimated, providing a more comprehensive analysis of the variants present than would be obtained by sequencing alone. Sequences from many individuals showed frequent amino acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the immunodominant loop. The rates of synonymous and nonsynonymous nucleotide substitution in the region of this and flanking regions indicate that strong positive selection for amino acid change is operating in the generation of antigenic diversity.     

Molecular Genetics of Herpes Simplex Virus II. Mapping of the Major Viral Glycoproteins and of the Genetic Loci Specifying the Social Behavior of Infected Cells

We have mapped the location in herpes simplex virus (HSV) DNA of (i) three mutations at different loci (syn loci) which alter the social behavior of infected cells from clumping of rounded cells to polykaryocytosis, (ii) a mutation which determines the accumulation of one major glycoprotein [VP8.0(C(2))], and (iii) the sequences encoding four major virus glycoproteins [VP8.0(C(2)), VP7(B(2)), VP8.5(A), and VP19E(D(2))]. The experimental design and results were as follows. (i) Analysis of HSV-1 x HSV-2 recombinants showed that the sequences encoding the VP19E(D(2)) glycoprotein map in the S component, whereas the sequences encoding the other three major glycoproteins are in two locations in the L component of HSV DNA. The templates specifying the HSV-1 and HSV-2 glycoprotein VP8.0(C(2)) appear not to be colinear; we isolated recombinants specifying glycoproteins comigrating in sodium dodecyl sulfate-polyacrylamide gels with VP8.0(C(2)) of both HSV-1 and HSV-2. (ii) Marker rescue of a ts mutant defective in accumulation of glycoprotein VP7(B(2)) showed that the mutation maps within a region containing the sequences encoding that glycoprotein. (iii) Marker transfer experiments involving transfection of rabbit skin cells with donor HSV-1(F) DNA and fragments from several donor strains causing fusion of Vero or both Vero and HEp-2 cells revealed the existence of three syn loci specifying the social behavior of cells and one locus (Cr) determining the accumulation of glycoprotein VP8.0(C(2)). The Cr locus maps to the right of the template specifying VP8.0(C(2)) glycoprotein. Loci syn 1 and syn 2 map at or near the Cr locus but can be segregated from it. Locus syn 3 maps at or near the template specifying glycoproteins VP7(B(2)) and VP8.5(A). The expression of mutations in the syn 1 and syn 3 loci appear to be cell type dependent, in that recombinants with these mutations fuse Vero cells but not HEp-2 cells. Recipients of the syn 2 locus or of both syn 2 and syn 1 loci fuse both Vero and HEp-2 cells.     

Improving accuracy of protein contact prediction using balanced network deconvolution

Residue contact map is essential for protein three‐dimensional structure determination. But most of the current contact prediction methods based on residue co‐evolution suffer from high false‐positives as introduced by indirect and transitive contacts (i.e., residues A–B and B–C are in contact, but A–C are not). Built on the work by Feizi et al. (Nat Biotechnol 2013; 31:726–733), which demonstrated a general network model to distinguish direct dependencies by network deconvolution, this study presents a new balanced network deconvolution (BND) algorithm to identify optimized dependency matrix without limit on the eigenvalue range in the applied network systems. The algorithm was used to filter contact predictions of five widely used co‐evolution methods. On the test of proteins from three benchmark datasets of the 9th critical assessment of protein structure prediction (CASP9), CASP10, and PSICOV (precise structural contact prediction using sparse inverse covariance estimation) database experiments, the BND can improve the medium‐ and long‐range contact predictions at the L/5 cutoff by 55.59% and 47.68%, respectively, without additional central processing unit cost. The improvement is statistically significant, with a P‐value < 5.93 × 10^?3 in the Student's t‐test. A further comparison with the ab initio structure predictions in CASPs showed that the usefulness of the current co‐evolution‐based contact prediction to the three‐dimensional structure modeling relies on the number of homologous sequences existing in the sequence databases. BND can be used as a general contact refinement method, which is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/BND/ . Proteins 2015; 83:485–496. © 2014 Wiley Periodicals, Inc.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.     

Detection of multiple β-casein (CASB) alleles by amplification created restriction sites (ACRS)

Direct sequencing of polymerase chain reaction (PCR) amplified DNA has been used to detect the DNA sequences for bovine beta-casein (CASB) A3 and B variants. Based on these sequences we have designed primers which create allele-specific restriction sites in the PCR product. Restriction analysis of PCR product generated in one reaction enable us to identify the A1, A2, A3 and B alleles of CASB rapidly without the use of radioactivity.     

Cleavage site mutations in the encephalomyocarditis virus P3 region lethally abrogate the normal processing cascade.

Site-specific mutations within the proteinase 3C-dependent P3 region cleavage sequences of encephalomyocarditis virus have been constructed. The mutations altered the normal QG cleavage site dipeptide pairs of the 2C/3A, 3A/3B, 3B/3C, and 3C/3D junctions into QV, QC, QF, QY, and RG sequences. When translated in vitro in the context of full-length viral polyproteins, all mutations blocked endogenous 3C-mediated processing at their engineered sites and produced stable forms of the expected viral P3 precursors that were also resistant to cleavage by exogenously added recombinant 3C. Relative to wild-type viral sequences, each mutant form of P3 had a somewhat different ability to mediate overall polyprotein processing. Mutations at the 2C/3A, 3A/3B, and 3B/3C sites, for example, were generally less impaired than 3C/3D mutations, when the cleavage reactions were quantitated with cotranslated L-P1-2A precursors. A notable exception was mutant 3B3C(QG-->RG), which proved far less active than sibling mutants 3B3C(QG-->QF) and 3B3C(QG-->QV), a finding that possibly implicates this segment in the proper folding of an active 3C. When transfected into HeLa cells, all mutant sequences were lethal, presumably because of the reduced L-P1-2A processing levels or reduced RNA synthesis capacity. However, when specifically tested for the latter activity, all mutations except those at the 3C/3D cleavage site were indeed able to initiate and perpetuate viral RNA replication in transfected cells, albeit to RNA accumulation levels lower than those produced by wild-type sequences. The transfection effects could be mimicked with cell-free synthesized proteins, in that translation samples containing locked 3CD polymerase precursors were catalytically inactive in poly(A)-oligo(U)-dependent assays, while all other mutant processing samples initiated detectable RNA synthesis. Surprisingly, not only did the 3B/3C mutant sequences prove capable of directing RNA synthesis, but the viral RNA thus synthesized could be immunolabeled and precipitated with 3C-specific monoclonal antibody reagents, indicating an unexpected covalent attachment of the proteinase to the RNA product whenever this cleavage site was blocked.