UV-filter pollution: current concerns and future prospects.

UV-filters are widely used in cosmetics and personal care products to protect users' skin from redamage caused by ultraviolet (UV) radiation from the sun. Globally, an estimated 16,000 to 25,000 tonnes of products containing UV-filters were used in 2014 with modern consumption likely to be much higher. Beyond this use in cosmetics and personal care products, UV-filters are also widely used to provide UV-stability in industrial products such as paints and plastics. This review discusses the main routes by which UV-filters enter aquatic environments and summarises the conclusions of studies from the past 10 years that have investigated the effects of UV-filters on environmentally relevant species including corals, microalgae, fish, and marine mammals. Safety data regarding the potential impact of UV-filters on human health are also discussed. Finally, we explore the challenges surrounding UV-filter removal and research on more environmentally friendly alternatives to current UV-filters.

Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans.

Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.

Canine recommended breed weight ranges are not a good predictor of an ideal body condition score.

Breed-specific ideal bodyweight range information is widely used by dog owners and breeders as a guideline to ensure animals are within a healthy weight range. Body Condition Scoring, a method used by veterinarians to assess an animal's overall shape with regard to weight is considered to be an excellent method to determine an animal's overall body condition; these values, however, do not always correspond to published weight ranges. Here, the weight, neuter status, age and a nine-point Body Condition Score of a population of 140 purebred dogs were recorded and subsequently analysed to determine whether bodyweight was an effective predictor for Body Condition Scores. This comparison indicated that published recommended, breed-specific body weight ranges are not a good predictor for an ideal BCS and as such, guidelines for owners and breeders need to be systematically reviewed.

Time-lapse embryo imaging and morphokinetic profiling: Towards a general characterisation of embryogenesis.

In vitro fertilisation is an effective method of assisted reproductive technology in both humans and certain non-human animal species. In most species, specifically, in humans and livestock, high in vitro fertilisation success rates are achieved via the transfer of embryos with the highest implantation and subsequent developmental potential. In order to reduce the risk of multiple gestation, which could be a result of the transfer of several embryos per cycle, restrictive transfer policies and methods to improve single embryo selection have been implemented. A non-invasive alternative to standard microscopic observation of post-fertilisation embryo morphology and development is time-lapse technology; this enables continuous, uninterrupted observation of embryo development from fertilisation to transfer. Today, there are several time-lapse devices that are commercially available for clinical use, and methods in which time-lapse could be used to improve embryology are continually being assessed. Here we review the use of time-lapse technology in the characterisation of embryogenesis and its role in embryo selection. Furthermore, the prospect of using this technology to identify aneuploidy in human embryos, as well as the use of time-lapse to improve embryological procedures in agriculturally important species such as the pig and cow are discussed.

Cryopreservation of animal oocytes and embryos: Current progress and future prospects.

Cryopreservation describes techniques that permit freezing and subsequent warming of biological samples without loss of viability. The application of cryopreservation in assisted reproductive technology encompasses the freezing of gametes, embryos, and primordial germ cells. Whilst some protocols still rely on slow-freezing techniques, most now use vitrification, or ultra-rapid freezing, for both oocytes and embryos due to an associated decreased risk of damage caused by the lack of ice crystal formation, unlike in slow-freezing techniques. Vitrification has demonstrated its use in many applications, not only following IVF procedures in human embryology clinics but also following in vitro production of embryos in agriculturally important, or endangered animal species, before embryo transfer. Here, we review the various cryopreservation and vitrification technologies that are used in both humans and other animals and discuss the most recent innovations in vitrification with a particular emphasis on their applicability to animal embryology.

Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the appropriate induction of dauer larvae development within growing populations is likely to be a primary determinant of genotypic fitness. The underlying genetic architecture of natural genetic variation in dauer formation has, however, not been thoroughly investigated. Here, we report extensive natural genetic variation in dauer larvae development within growing populations across multiple wild isolates. Moreover, bin mapping of introgression lines (ILs) derived from the genetically divergent isolates N2 and CB4856 reveals 10 quantitative trait loci (QTLs) affecting dauer formation. Comparison of individual ILs to N2 identifies an additional eight QTLs, and sequential IL analysis reveals six more QTLs. Our results also show that a behavioural, laboratory-derived, mutation controlled by the neuropeptide Y receptor homolog npr-1 can affect dauer larvae development in growing populations. These findings illustrate the complex genetic architecture of variation in dauer larvae formation in C. elegans and may help to understand how the control of variation in dauer larvae development has evolved.

Thermal variation reveals natural variation between isolates of Caenorhabditis elegans.

The free-living nematode Caenorhabditis elegans is distributed globally and found in many varied habitats. However, in comparison to our understanding of the genetics of the species, little is known about natural variation and many major life history traits appear to show only limited differences between isolates. Here we show that temperature affects the lifetime fecundity and the reproductive timing of C. elegans and that there is a genotype by environment interaction, with isolates varying in how lifetime fecundity changes with temperature. We show that the lower lifetime fecundity observed at higher temperatures is primarily due to a reduction in the number of functional sperm. Further, isolates vary in their lifetime fecundity because of inter-isolate differences in this effect of temperature on the number of functional sperm.

The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.

RATIONALE: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. OBJECTIVES AND METHODS: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. RESULTS: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. CONCLUSIONS: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus.

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.

Physical mapping of the brain and ovarian aromatase genes in the Nile Tilapia, Oreochromis niloticus, by fluorescence in situ hybridization.

Cytochrome P450-aromatase enzyme (CYP19), which catalyses the conversion of androgens to oestrogens, is critical in ovarian differentiation and hence in the sex differentiation pathways of non-mammalian vertebrates. As in other fish species, distinct ovarian and brain aromatase genes have been identified in the Nile Tilapia, Oreochromis niloticus. Here we demonstrate by in situ hybridization that the two aromatase genes of this species are present on different chromosomes and that neither are located on the sex chromosomes. Hence, the aromatase genes are not the primary sex determination genes in O. niloticus.

Molecular-cytogenetic analysis reveals sequence differences between the sex chromosomes of Oreochromis niloticus: evidence for an early stage of sex-chromosome differentiation.

Sex determination in the Nile tilapia, Oreochromis niloticus, is primarily genetic, with XX females and XY males. A candidate sex-determining region in the terminal region of the largest chromosome pair has been identified by analysis of meiotic chromosomes. This region shows an inhibition of pairing and synapsis in the XY genotype, but not in XX or YY genotypes, suggesting that recombination is inhibited. Here we show that chromosome microdissection and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR) can be used to produce in situ hybridization probes to this largest pair of O. niloticus chromosomes. Furthermore, analysis of the comparative hybridization of X and Y chromosome-derived probes to different genotypes provides the first demonstration that sequence differences exist between the sex chromosomes of O. niloticus. This provides further support for the theory that this chromosome pair is related to sex determination and further suggests that the sex chromosomes are at a very early stage of divergence.

Karyotype evolution in Tilapia: mitotic and meiotic chromosome analysis of Oreochromis karongae and O. niloticus x O. karongae hybrids.

The karyotype of Oreochromis species is considered to be highly conserved, with a diploid chromosome complement of 2n = 44. Here we show, by analysis of mitotic and meiotic chromosomes, that the karyotype of O. karongae, one of the Lake Malawi 'chambo' species, is 2n = 38. This difference in chromosome number does not prevent the production of inter-specific hybrids between O. niloticus (2n = 44) and O. karongae (2n = 38). Analysis of the meiotic chromosomes of the O. niloticus x O. karongae hybrids indicates that three separate chromosome fusion events have occurred in O. karongae. Comparison of the O. karongae and O. niloticus karyotypes suggests that these consist of one Robertsonian fusion and two fusions of a more complex nature.

Early origins of the X and Y chromosomes: lessons from tilapia.

Differentiated sex chromosome pairs in diverse species display certain common characteristics, normally comprising one largely heterochromatic genetically inactive chromosome and one euchromatic genetically active chromosome (e.g. the mammalian Y and X respectively). It is widely accepted that dimorphic sex chromosomes evolved from homologous pairs of autosomes. Although the exact mechanisms through which the pair diverged are not fully understood, an initial suppression of recombination in the sex-determining region is required by all of the major theories. Here we address the question of the mechanism by which this initial suppression of recombination occurs. Our model postulates that the stochastic, de novo accumulation of heterochromatin in the sex determining region can delay pairing of the sex chromosomes in meiosis, resulting in a decrease in recombination. Data to support this model is presented from the cichlid fish, Oreochromis niloticus. Although such a decrease would in most circumstances be evolutionarily disadvantageous, if the region concerned included the major sex determining gene and other gene(s) with sex-specific functions, then this would be selectively advantageous and could trigger the process(es) which, ultimately, lead to the differentiation of the sex chromosomes.

AMPA receptor function is altered in GLUR2-deficient mice.

The GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor determines many of the biophysical properties of native AMPA receptors, including Ca++ permeability. Genetically engineered mice unable to edit the Q to R site of the GluR2 subunit die within 3 wk postpartum, presumably due to toxicity associated with enhanced Ca++ influx through AMPA receptors. In contrast, disruption of the gene encoding GluR2 is not necessarily lethal. The objective of this study was to explore potential mechanisms that permit survival of GluR2 (-/-) mice despite AMPA receptors that are highly Ca++ permeable. Whole-cell, patch-clamp recording of AMPAreceptor responses in cortical pyramidal cells revealed that the kinetics of recovery from desensitization were significantly slower for receptors from GluR2 (-/-) mice compared to receptors from GluR2 (+/+) mice. The recovery time constants for AMPA receptors from GluR2 (-/-) and GluR2 (+/+) mice were 109.8 +/- 17 ms and 54.4 +/- 7.1 ms, respectively. The slower recovery kinetics would be expected to reduce Ca++ influx during repetitive stimulation. Because both RNA editing at the R/G site and alternative splicing of the flip and flop module affect AMPA receptor desensitization recovery rates, the possibility that these mechanisms were changed in GluR2 (-/-) mice was investigated. On a macroscopic level, neither editing nor splicing of the GluR-1, 3 or 4 subunits were changed in GluR2 (-/-) mice compared to GluR2 (+/+) mice. In summary, an increase in the time constant for recovery from desensitization may contribute to the ability of GluR2 (-/-) to survive.

5-hydroxytryptamine2A receptor inverse agonists as antipsychotics.

We have used a cell-based functional assay to define the pharmacological profiles of a wide range of central nervous system active compounds as agonists, competitive antagonists, and inverse agonists at almost all known monoaminergic G-protein-coupled receptor (GPCR) subtypes. Detailed profiling of 40 antipsychotics confirmed that as expected, most of these agents are potent competitive antagonists of the dopamine D2 receptor. Surprisingly, this analysis also revealed that most are potent and fully efficacious 5-hydroxytryptamine (5-HT)2A receptor inverse agonists. No other molecular property was shared as universally by this class of compounds. Furthermore, comparisons of receptor potencies revealed that antipsychotics with the highest extrapyramidal side effects (EPS) liability are significantly more potent at D2 receptors, the EPS-sparing atypical agents had relatively higher potencies at 5-HT2A receptors, while three were significantly more potent at 5-HT2A receptors. Functional high-throughput screening of a diverse chemical library identified 530 ligands with inverse agonist activity at 5-HT2A receptors, including several series of compounds related to known antipsychotics, as well as a number of novel chemistries. An analog of one of the novel chemical series, AC-90179, was pharmacologically profiled against the remaining monoaminergic GPCRs and found to be a highly selective 5-HT2A receptor inverse agonist. The behavioral pharmacology of AC-90179 is characteristic of an atypical antipsychotic agent.

Determinants of agonist binding affinity on neuronal nicotinic receptor beta subunits.

The alpha and beta subunits of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are thought to contribute "principal" and "complementary" components to the agonist binding site, respectively. At least six loops of amino acid sequence (A, B, and C from alpha; D, E, and F from beta) are involved. We demonstrated previously that receptors containing the beta2 subunit had consistently higher affinities for a variety of agonists than beta4-containing receptors. For example, the affinity of the alpha2beta2 receptor for epibatidine, ACh, nicotine, and dimethylphenylpiperazinium (DMPP) exceeds that of alpha2beta4 by 9-, 61-, 87-, and 120-fold, respectively. Using saturation and competition analysis of receptors formed by chimeric beta subunits coexpressed with alpha2 in Xenopus laevis oocytes, we have now identified sequence segment 54-63 (corresponding to loop D) as a major determinant of affinity for epibatidine, ACh, nicotine, and DMPP. We then analyzed a series of mutant beta2 subunits in which each residue that differs between beta2 and beta4 in this region was changed from what occurs in beta2 to what occurs in beta4. The N55S, V56I, and E63T mutations each resulted in a loss of affinity for ACh and nicotine of 3- to 4-fold, whereas the T59K mutation resulted in a 7-fold loss of ACh and nicotine affinity. These mutations had little or no effect on epibatidine and DMPP affinity. The positive charge introduced by the T59K mutation does not appear to underlie loss of agonist affinity, because a similar loss of affinity was observed when a negative charge (T59D) was introduced at this position.

Sex determination in the parasitic nematode Strongyloides ratti.

The parasitic nematode Strongyloides ratti reproduces by both parthenogenesis and sexual reproduction, but its genetics are poorly understood. Cytological evidence suggests that sex determination is an XX/XO system. To investigate this genetically, we isolated a number of sex-linked DNA markers. One of these markers, Sr-mvP1, was shown to be single copy and present at a higher dose in free-living females than in free-living males. The inheritance of two alleles of Sr-mvP1 by RFLP analysis was consistent with XX female and XO male genotypes. Analysis of the results of sexual reproduction demonstrated that all progeny inherit the single paternal X chromosome and one of the two maternal X chromosomes. Therefore, all stages of the S. ratti life cycle, with the exception of the free-living males, are XX and genetically female. These findings are considered in relation to previous analyses of S. ratti and to other known sex determination systems.

AA.AG@helix.ends: A:A and A:G base-pairs at the ends of 16 S and 23 S rRNA helices.

This study reveals that AA and AG oppositions occur frequently at the ends of helices in RNA crystal and NMR structures in the PDB database and in the 16 S and 23 S rRNA comparative structure models, with the G usually 3' to the helix for the AG oppositions. In addition, these oppositions are frequently base-paired and usually in the sheared conformation, although other conformations are present in NMR and crystal structures. These A:A and A:G base-pairs are present in a variety of structural environments, including GNRA tetraloops, E and E-like loops, interfaced between two helices that are coaxially stacked, tandem G:A base-pairs, U-turns, and adenosine platforms. Finally, given structural studies that reveal conformational rearrangements occurring in regions of the RNA with AA and AG oppositions at the ends of helices, we suggest that these conformationally unique helix extensions might be associated with functionally important structural rearrangements.

Structural studies of the tRNA domain of tmRNA.

tmRNA is a small, stable prokaryotic RNA. It rescues ribosomes that have become stalled during the translation of mRNA fragments lacking stop codons, or during periods of tRNA scarcity. It derives its name from the presence of two separate domains, one that functions as a tRNA, and another that serves as an mRNA. We have carried out modeling and transient electric birefringence studies to determine the angle between the acceptor stem and anticodon stem of the tRNA domain of Eschericia coli tmRNA. The results of the modeling studies yielded an interstem angle of 110 degrees, in agreement with the lower end of the range of angles (111 degrees -137 degrees ) determined experimentally for various solution conditions. The range of experimental angles is greater than the angles observed for any of the tRNA crystal structures, in line with the presence of a shortened D stem. The secondary structure of the tRNA domain is conserved for all known tmRNA sequences, so we propose that the angle is also conserved. These results also suggest that the region of tmRNA between P2a and P2b may interact with the decoding site of the ribosome.