Neurochemical Interactions of Competitive N-Methyl-D-Aspartate Antagonists with Dopaminergic Neurotransmission and the Cerebellar Cyclic GMP System: Functional Evidence for a Phasic Glutamatergic Control of the Nigrostriatal Dopaminergic Pathway

Direct intrastriatal injection of N-methyl-D-aspartate (NMDA; 100 micrograms/rat) increased striatal dopamine (DA) release in vivo. However, parenteral administration of (+/-)-3-(2-carboxypiperizin-4-yl)propyl-1-phosphonic acid (CPP) and cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS-19755) did not alter DA metabolism and release in several brain regions in the rat and mouse. Intracerebroventricular administration of the competitive NMDA antagonists CPP, CGS-19755, 2-amino-5-phosphonopentanoate, and 2-amino-7-phosphonoheptanoate did not alter rat striatal DA metabolism and release but profoundly reduced cerebellar cyclic GMP (cGMP) levels in the same animals. CPP and CGS-19755 decreased basal cerebellar cGMP levels in the mouse with ED50 values of 6 and 1 mg/kg, i.p., respectively. CPP antagonized the harmaline-induced increases in cGMP levels with an ED50 value of 5.0 mg/kg, i.p. CPP (25 mg/kg, i.p.) also decreased basal cGMP levels in mouse cerebellum for up to 3 h, a result suggesting brain bioavailability and a long duration of NMDA receptor antagonism in vivo. These contrasting patterns suggest that NMDA receptors exert a tonic excitatory tone on the guanine nucleotide signal transduction pathway in the cerebellum while exerting a phasic control over nigrostriatal dopaminergic neurotransmission. These results also indicate that competitive NMDA antagonists, unlike phencyclidine receptor agonists, may not mediate biochemical and behavioral effects via dopaminergic mechanisms.     

Growth-regulated proteins and neuronal plasticity

Growth-regulated proteins (GRPs) of the neuron are synthesized during outgrowth and regeneration at an increased rate and enriched in nerve growth cones. Therefore, they can be used to some degree as markers of neurite growth. However, these proteins are not unique to the growing neuron, and their properties are not known sufficiently to assign them a functional and/or causal role in the mechanisms of outgrowth. During synaptogenesis, GRPs decrease in abundance, and growth cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release. However, there is a stage during which growth cone and synaptic properties overlap to some degree. We propose that it is this overlap and its continuation that allow for synaptic plasticity in developing and adult nervous systems. We also propose a hypothesis involving (a) trophic factor(s) that might explain the regulation of synaptic sizes and collateral sprouting. Some GRPs, especially GAP43/B50/pp46/F1, are more prominent in adult brain regions of high plasticity, and they undergo change, such as phosphorylation, during long-term potentiation (LTP). Without precise functional knowledge of GRPs, it is impossible to use changes in such proteins to explain the plasticity mechanism. However, changes in these "growth markers" are likely to be an indication of sprouting activity, which would explain well the various phenomena associated with plasticity and learning in the adult. Thus, plasticity and memory may be viewed as a continuation of the developmental process into adulthood.     

A multi-split mapping algorithm for circular RNA,splicing, trans-splicing and fusion detection

Numerous high-throughput sequencing studies have focused on detecting conventionally spliced mRNAs in RNA-seq data. However, non-standard RNAs arising through gene fusion, circularization or trans-splicing are often neglected. We introduce a novel, unbiased algorithm to detect splice junctions from single-end cDNA sequences. In contrast to other methods, our approach accommodates multi-junction structures. Our method compares favorably with competing tools for conventionally spliced mRNAs and, with a gain of up to 40% of recall, systematically outperforms them on reads with multiple splits, trans-splicing and circular products. The algorithm is integrated into our mapping tool segemehl (http://www.bioinf.uni-leipzig.de/Software/segemehl/).     

Effect of high-carbon dioxide atmospheres on infestations of apple maggot (Diptera: Tephritidae) in apples

Short-term storage regimens containing elevated atmospheres of carbon dioxide (CO2) were evaluated for their ability to disinfest newly harvested 'McIntosh' apples of apple maggot, Rhagoletis pomonella (Walsh). Infested fruits containing newly laid eggs were either placed directly into the high-CO2 atmosphere at 10 degrees C to expose this life stage, or else held first for 7 d at room temperature, to allow development to the neonate larval stage. Treatment combinations consisted of three different CO2 levels (10.6, 14.9, and 19.0% CO2) and two periods of exposure (7 and 14 d). Apple maggot eggs subjected to the treatments always exhibited some survival, which was lower for the 14-d than the 7-d exposure periods. In contrast, newly hatched larvae were less able to survive the treatments. The 7-d exposure allowed low levels of survival of this life stage, but virtually none survived the 14-d exposure period. To determine the age at which eggs become more susceptible to high-CO2 atmospheres, infested fruits containing eggs three or 3d old were submitted to a 14-d exposure to 19.0% CO2. Survival of 3-d old eggs was similar to that of eggs exposed at an age of 1 d or less, but this dropped to near zero for 5-d old eggs, indicating an increase in susceptibility sometime during the 3-5-d age range. Fruits exposed to 19.0% CO2 for 14 d were significantly firmer than untreated fruits. No apparent browning, internal breakdown or other fruit defects were detected in any of the treatments.     

A Spotlight on Preschool: The Influence of Family Factors on Children’s Early Literacy Skills

Rationale

Phonological awareness, letter knowledge, oral language (including sentence recall) and rapid automatised naming are acknowledged within-child predictors of literacy development. Separate research has identified family factors including socio-economic status, parents’ level of education and family history. However, both approaches have left unexplained significant amounts of variance in literacy outcomes. This longitudinal study sought to improve prospective classification accuracy for young children at risk of literacy failure by adding two new family measures (parents’ phonological awareness and parents’ perceived self-efficacy), and then combining the within-child and family factors.

Method

Pre-literacy skills were measured in 102 four year olds (46 girls and 56 boys) at the beginning of Preschool, and then at the beginning and end of Kindergarten, when rapid automatised naming was also measured. Family factors data were collected at the beginning of Preschool, and children’s literacy outcomes were measured at the end of Year 1 (age 6–7 years).

Results

Children from high-risk backgrounds showed poorer literacy outcomes than low-risk students, though three family factors (school socio-economic status, parents’ phonological awareness, and family history) typically accounted for less Year 1 variance than the within-child factors. Combining these family factors with the end of Kindergarten within-child factors provided the most accurate classification (i.e., sensitivity = .85; specificity = .90; overall correct = .88).

Implications

Our approach would identify at-risk children for intervention before they began to fail. Moreover, it would be cost-effective because although few at-risk children would be missed, allocation of unnecessary educational resources would be minimised.     

Tn502 and Tn512 Are res Site Hunters That Provide Evidence of Resolvase-Independent Transposition to Random Sites

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.It is widely recognized that mobile genetic elements contribute to genome plasticity and have been a driving force in the emergence and spread of resistance determinants within and between bacterial species; their impact is ongoing (10, 51). Significant among these elements are various classes of plasmids, transposons, and integrons which may lack resistance determinants or carry one or multiple determinants. Resistance determinants that have become globally dispersed in environmental and clinically significant bacteria include mercury(II) resistance (2, 17), evident even in ancient bacteria (27), and antibiotic resistance, which has increased in dominance since the advent of the antibiotic era (23, 40).This paper concerns the mercury resistance (mer) transposons Tn502 and Tn512, whose sequence organization and transpositional behavior show that they are new members of a family of elements exemplified by the mer transposon Tn5053 (22). These elements are closely related to those in the Tn402 family, which contain an integron (intI) recombination system (14, 36). Members of the two families differ in the positions of the mer or intI determinants (modules) near one end of the transposition (tni) module. The latter module contains four genes (tniABQR), and the entire transposon is bounded by 25-bp inverted-repeat termini (IRi and IRt). TniA, TniB, and TniQ are required to form the transpositional cointegrate, which is then resolved by the action of TniR (a serine resolvase) on a resolution (res) sequence located between tniR and tniQ (22). The transposon in its new location is flanked by 5-bp direct repeats (DRs) (20, 22). TniA, which contains a D,D(35)E transposase catalytic motif, is thought to function cooperatively with TniB, a putative nucleotide-binding protein, as the active TniAB transposase (21, 36). Studies of TniA conducted in vitro show binding to the IRs and to additional 19-bp repeat sequences that make up the complex termini of the transposon (21). The precise role of TniQ is unknown.An unexpected and unique feature of Tn5053 and Tn402 is that they depend on externally coded accessory functions for efficient transposition, namely, a res site served by a cognate resolvase (25). As a consequence, these transposons exhibit a strong transpositional bias for some target res sites (20, 25, 26) and have aptly been described as “res site hunters” (25). One such efficient interaction involves the res-ParA multimer resolution system of plasmid RP1 (IncPα); other plasmid- or transposon-encoded systems are less efficient or are refractory. Although the role of the external resolvase remains obscure, its capacity to bind to its cognate res is an essential requirement whereas its catalytic activity is not (20). For each interaction system, the target sites typically cluster in a single part of res but not necessarily within the same subregion and, on occasion, can lie in the vicinity of res. Typically, the transposon is in a single orientation with IRi closest to the resolvase gene. In one study, Tn402 clustered at two target sites, one within res and one nearby, and the orientations were different at the two sites (20).The experimentally observed target preference described above also occurs in natural associations of Tn5053/Tn402-like elements and became evident on sequencing class 1 integrons, which were often found positioned close to different res-resolvase gene regions (6, 20, 25). Most Tn402 family elements are comprised of an intI module that is flanked on the left by IRi and on the right by a 3′ conserved sequence (3′-CS) (13). In others, a remnant tni gene cluster may be present instead of the 3′-CS, and IRt occurs at the right flank. The structure of the latter category of integrons strongly indicated that they are defective transposons that were presumably capable of relocation provided that tni functions were supplied in trans (6, 32). The movement of In33 (Tn2521) from a chromosomal to a plasmid location appears to have been such an in trans event (30, 42), and others involving In0 and In2 are demonstrated in this study. In contrast, the integrons that lack the IRt end appear to be nonmobile remnants of Tn402-like transposons; they belong to several lineages, including those in which the incurred deletions are attributable to acquired insertion sequences (6). More recently, intact Tn5053/Tn402-like transposons and class 1 integrons have increasingly been detected in the res-parA region of IncP plasmids (39), which are arguably the most promiscuous of known plasmids (50). These various experimental and natural interactions provide insight into the dispersal pathways possible for Tn5053/Tn402-like elements.The res-hunting attribute is a striking feature that is experimentally supported by studies of four family members (namely, Tn5053 [22, 25], Tn402 [20, 26], and in this study, Tn502 [48] and Tn512). Another facet of the transposition of Tn502 is explored here. It concerns the observation that loss of the preferred par target region in RP1 does not abolish transposition of Tn502 (48), contrary to the finding with Tn5053 (25, 26) and, in this study, Tn512. The continued, low-frequency transposition of Tn502 involved at least three dispersed locations (48); however, nothing is known about the nature of these sites or about the features and requirements of the transposition process. Here we address these issues and uncover the existence of an alternative, par-independent pathway that is employed by Tn502 and is available to Tn512 under some circumstances. The study also provides information on the roles of the TniR and host (RecA) recombination systems in the resolution of transpositional cointegrates and on the ability of the par-independent transposition pathway to generate plasmid deletions.     

Multivariate phenotype analysis enables genome-wide inference of mammalian gene function

The function of the majority of genes in the human and mouse genomes is unknown. Investigating and illuminating this dark genome is a major challenge for the biomedical sciences. The International Mouse Phenotyping Consortium (IMPC) is addressing this through the generation and broad-based phenotyping of a knockout (KO) mouse line for every protein-coding gene, producing a multidimensional data set that underlies a genome-wide annotation map from genes to phenotypes. Here, we develop a multivariate (MV) statistical approach and apply it to IMPC data comprising 148 phenotypes measured across 4,548 KO lines.There are 4,256 (1.4% of 302,997 observed data measurements) hits called by the univariate (UV) model analysing each phenotype separately, compared to 31,843 (10.5%) hits in the observed data results of the MV model, corresponding to an estimated 7.5-fold increase in power of the MV model relative to the UV model. One key property of the data set is its 55.0% rate of missingness, resulting from quality control filters and incomplete measurement of some KO lines. This raises the question of whether it is possible to infer perturbations at phenotype–gene pairs at which data are not available, i.e., to infer some in vivo effects using statistical analysis rather than experimentation. We demonstrate that, even at missing phenotypes, the MV model can detect perturbations with power comparable to the single-phenotype analysis, thereby filling in the complete gene–phenotype map with good sensitivity.A factor analysis of the MV model’s fitted covariance structure identifies 20 clusters of phenotypes, with each cluster tending to be perturbed collectively. These factors cumulatively explain 75% of the KO-induced variation in the data and facilitate biological interpretation of perturbations. We also demonstrate that the MV approach strengthens the correspondence between IMPC phenotypes and existing gene annotation databases. Analysis of a subset of KO lines measured in replicate across multiple laboratories confirms that the MV model increases power with high replicability.

The function of the majority of genes in the human and mouse genomes is unknown, and illuminating this "dark genome" is a major challenge for the biomedical sciences. This study shows that multi-dimensional phenotypes from single-gene knockout mouse lines can be analysed at a genome-wide scale both to increase power and infer missing phenotypes.