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.

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.

Phenylalanine transfer RNA: molecular dynamics simulation.

Yeast phenylalanine transfer RNA was subjected to a 12-picosecond molecular dynamics simulation. The principal features of the x-ray crystallographic analysis are reproduced, and the amplitudes of atomic displacements appear to be determined by the degree of exposure of the atoms. An analysis of the hydrogen bonds shows a correlation between the average length of a bond and the fluctuation in that length and reveals a rocking motion of bases in Watson-Crick guanine X cytosine base pairs. The in-plane motions of the bases are generally of larger amplitude than the out-of-plane motions, and there are correlations in the motions of adjacent bases.

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.

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.

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors.

Active transport of ions across membranes: energetic role of electrostatics and binding site asymmetry.

The active transport of ions across a membrane by an ATP-driven electrogenic ion pump is often described by an 'alternate access' model. The position of the binding site is assumed to be unchanged as the binding cavity opens alternatively to the uptake and discharge sides of the membrane. The ion binding affinity is higher on the uptake side of the membrane than on the discharge side. This difference in affinities is related to the maximum transport rate and to the efficiency with which ATP hydrolysis is coupled to active transport. Here we examine the electrostatic contribution to binding affinities, using a simple geometry for a model membrane-protein system, a continuum dielectric approximation, and a numerical method to calculate binding energy as a function of the binding site location. If the binding site is located asymmetrically, being further from the uptake side of the membrane than from the discharge side, there is a significant difference in binding free energy between the uptake and discharge states. This asymmetry can produce differences in affinities that are consistent with those measured for biological active transport systems. These results may account for the observed asymmetric location of the calcium binding site in the calcium ATPases from sarcoplasmic reticulum and from the plasma membrane. Electrostatic energy differences associated with binding site asymmetry may be a general feature of electrogenic transmembrane ion pumps.

Molecular mechanics model of supercoiled DNA.

We describe a pseudo-atomic model of supercoiled DNA. Each base-pair of the DNA is represented in the model by three particles placed in a plane. The particle triplets are stacked to model stacked base-pairs in double-helical DNA, and closed circular conformations are generated to investigate supercoiling. This model is less detailed than all-atom models, which are too computationally demanding to be used to study supercoiling. On the other hand, this model contains details at the base-pair level and is therefore more elaborate than elastomechanical models. A potential energy function is written in terms of a set of internal co-ordinates defined to resemble a limited number of helical parameters. The modeled helical parameters, helical twist, base-roll, tilt and rise, are the most important parameters of the global shape of DNA. Experimentally measured mechanical properties of DNA are used to define the forces holding the particles together. We then use a procedure incorporating energy minimization and molecular dynamics to locate low energy conformations of the model DNA. The model was found to behave very much like rubber-tubing and elastomechanical models. The conformations and the effects of supercoiling pressure (a number proportional to the degree to which the total twist of the DNA has been altered from its natural value) on these conformations are all very similar to those observed in the latter two models. We also used this model to examine the effects of supercoiling pressure, base-sequence and mechanical properties on the conformations and energies of five sequences. The sequences studied include models of naturally straight DNA and DNA with static or natural bends.

A quantitative model of the Escherichia coli 16 S RNA in the 30 S ribosomal subunit.

We use a computer-based protocol for automated structure refinement of large RNAs and ribonucleoproteins to propose a three-dimensional model for the Escherichia coli 16 S RNA in the 30 S ribosomal subunit along with the first quantitative estimates of the uncertainties in the model. Our models are based on the 16 S RNA secondary structure, the small angle neutron scatter map of 30 S proteins, tertiary RNA-RNA and RNA-protein contacts as suggested by cross-linking, chemical footprinting and other experimental studies, and electron microscopy data for the shape of the 30 S subunit and placement of 16 S RNA fragments, along with known motifs in RNA structure. In addition, some data on the interaction of the tRNAs/mRNA with the 16 S RNA were used to localize the active site. Since there are not enough structural data to derive a unique three-dimensional folding of the 16 S RNA, several different conformations can be generated to satisfy the experimental data. A set of seven models was refined to survey the range of acceptable conformations. These models were analyzed to deduce probable positions and orientations of the different helical segments that comprise the 16 S RNA in the Escherichia coli small subunit, and one consensus model from this set is presented here. An estimate of the reliability of our predicted structure is made using the variations between the models, and about 75% of 16 S RNA helical segments are localized to 15 A or less in their position in the small subunit. Our models show a distinct separation of the three major domains of the 16 S RNA. The 5' major domain and the central domain are clustered in the body of the 30 S subunit, whereas the 3' major domain is localized in the head of the subunit. Our modeling results are compared with models of the 16 S RNA proposed by other researchers, and are seen to be similar to the manually built models by Stern et al. and Brimacombe et al. with a few significant differences. The position of nucleotides implicated by footprinting and crosslinking data in tRNA and mRNA binding, and in subunit association are examined, and many of these sites are seen to lie along the 30 S subunit neck, cleft and the platform.

Exploring three-dimensional structures of the HIV-1 RNA/tRNALys3 initiation complex.

Human immunodeficiency virus type 1 (HIV-1) uses host tRNA as a primer for reverse transcription of its viral RNA. The 3' terminal 18 nucleotides of human tRNALys3 are complementary to the primer binding site on the viral RNA. A secondary structure model for the HIV-1 RNA/tRNALys3 initiation complex has been proposed that includes additional base-pairing between the tRNA and the HIV-1 RNA beyond the 18 nucleotides of the primer binding site. Included in these interactions is base-pairing between the anticodon of tRNALys3 and an A-rich loop in the HIV-1 secondary structure. The tRNA and HIV-1 RNA are significantly unfolded from their native structures in order to form the initiation complex proposed in this model. We have found several problems with the proposed secondary structure in our efforts to build a three-dimensional model that is compatible with it. The additional interactions between the tRNA and viral RNA cause the structure to be topologically knotted. This poses a problem for folding of the initiation complex and transcription by reverse transcriptase. We have also not been able to build any all-atom models based on known RNA structures that follow the secondary structure model in the extended tRNA/HIV-1 RNA complex. Finally, beyond the primer binding site interaction, subsequent biochemical and genetic studies have given further insight into the structure of the initiation complex. These results call into question some of the extended HIV-1 RNA/tRNA interactions that have been proposed.

Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site.

We propose a detailed three-dimensional model, with atomic detail, for the structure of the Escherichia coli 16 S rRNA decoding site in a complex with mRNA and the A and P-site tRNAs. Model building began with four primary assumptions: (1) A and P-site tRNA conformations are identical with those seen in the tRNA crystal structure; (2) A and P-site tRNAs adopt an S-type orientation upon binding mRNA in the ribosome; (3) A1492 and A1493 bind non-specifically to the mRNA through a series of hydrogen bonds; and (4) C1400 lies in close proximity to the P-site tRNA wobble base in order to satisfy a UV-induced photocrosslink formed between the two residues. We have models with both major groove and minor groove binding of the tRNA/mRNA complex to the decoding site RNA, and conclude that major groove binding is more likely. Both classes of models maintain structural features reported in the NMR structure of the A-site region of the decoding site RNA with bound paromomycin. We also present models for the tRNA/mRNA complex bound to the decoding site RNA in the presence of the aminoglycoside paromomycin. We discuss possible mechanisms for ribosomal proof reading and antibiotic disruption of this proofreading.

Three-dimensional placement of the conserved 530 loop of 16 S rRNA and of its neighboring components in the 30 S subunit.

Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved. Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding. Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits. The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base. The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure.

Movement of the decoding region of the 16 S ribosomal RNA accompanies tRNA translocation.

The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)(2) complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately 8 A toward the P-site. This implies that elongation factor G actively pushes both the decoding site and the mRNA/tRNA complex during translocation.

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.

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.

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.

Detailed molecular model of apolipoprotein A-I on the surface of high-density lipoproteins and its functional implications.

The major apolipoprotein (apo) A-I containing lipoprotein, high- density lipoprotein, is a negative risk factor for cardiovascular disease. An atomic resolution molecular model for lipid-associated apo A-I was recently proposed in which two apo A-I molecules are wrapped beltwise around a small discoidal patch of phospholipid bilayer. Because of its detailed predictions of lipid-associated apo A-I structure, this molecular belt model, if confirmed, provides a blueprint for understanding the molecular mechanisms of reverse cholesterol transport, and thus for the rational design of new classes of drugs for reversal of atherosclerosis and cardiovascular disease. The details and implications of the model are currently being explored by site-directed mutagenesis.

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.