Vocal Communication in Pigs: Who are Nursing Piglets Screaming at?

Vocalizations during competition among nursing piglets were studied to investigate their possible effects, functions and implications for welfare. In Expt 1, two experimental piglets in each of 14 litters were temporarily deprived of milk by covering their preferred teats on the sow's udder. These piglets spent more time away from their teats than two control piglets, and vocalized frequently in the 2 min before milk ejection. Frequency of vocalization showed no consistent change over time within nursings; nor did it change in successive nursings despite the fact that hunger presumably increased. In Expt 2, tape recordings of intense vocalizations (screams) produced by piglets competing at the udder were played to 22 litters while they were nursing; each litter was played its own recording, a recording from another litter and silence as a control. Of 51 nursings analysed, 14 were terminated without milk ejection, all during playbacks. When the sow did nurse successfully during a playback, nursing was shorter (138 s) than during the silent controls (179 s). Both these responses by the sow might be expected to advance the next nursing. Piglets rarely showed any apparent response to screaming either from their littermates or from the loudspeaker. These results suggest that the calls function mainly as a signal to the sow that some piglets are being excluded from the current nursing episode.     

Limitations of Time-Resolved Fluorescence Suggested by Molecular Simulations: Assessing the Dynamics of T?cell Receptor Binding Loops

Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops.     

Nano-mechanical Compliance of Müller Cells Investigated by Atomic Force Microscopy

It has been known that a single Müller cell displays a large variation in the cytoskeletal compositions along its cell body, suggesting different mechanical properties in different segments. Müller cells are thought to be involved in many retinal diseases such as retinoschisis, which can be facilitated by a mechanical stress. Thus, mapping of mechanical properties on localized nano-domains of Müller cells could provide essential information for understanding their structural functions in the retina and roles in their pathological progresses. Using Atomic Force Microscopy (AFM) - based bio-nano-mechanics, we have investigated the local variations of the mechanical properties of Müller cells in vitro. We have a particular interest in identifying elastic moduli in regions closer to three distinctive segments of the cells - process, endfoot, and soma. Using the modified spherical AFM probes, we were able to accurately determine mechanical properties, i.e., elastic moduli from the obtained force-distance curves. We found that the regions closer to soma were mechanically more compliant than regions closer to endfoot and process of Müller cells. We found that this lateral heterogeneity of the mechanical compliance within a single Müller cell is consistent with reports from other cell types. The local variation in mechanical compliances along a single Müller cell may support their diverse mechanical functions in the retina such as a soft mechanical embedding, mechanosensing, and neurotrophic functions for neurons.     

Self-association of lyophilized horse liver alcohol dehydrogenase.

The molecular weights of lyophilized and non-lyophilized horse liver alcohol dehydrogenase have been compared by quasi-elastic light scattering, and ultracentrifugation. Whereas the non-lyophilized enzyme has the expected molecular weight of 78 000, the lyophilized enz)me has an initial molecular weight of about 10(6) which increases with time by an endothermic process. This result shows that any physical measurement using lyophilized liver alcohol dehydrogenase to investigate the enzyme mechanism, which relies upon the molecular size, will be invalid.     

Modelling the dynamic biogeography of the wildcat: implications for taxonomy and conservation

There is still no clear consensus on how to relate geographical variation in the morphology and genetics of the globally widespread wildcat Felis silvestris to its taxonomy and systematics. Reconstructing the dynamic biogeography of the wildcat provides insight into how current geographical patterns of morphological and molecular variation may have developed. A geographical information system was used to infer climate-change influences using a deduced distribution model (DDM) to reconstruct the wildcat's geographical distribution at four points in time from the Last Glacial Maximum [LGM; 18 000 years before present ( bp )] until today. The DDM for 9000  bp , when mean global temperatures were 2 °C more than today, provides insight into how current global warming will affect the wildcat's distribution 50–100 years into future. Modelled distributions were assessed against known geographical barriers or unsuitable habitats, which may have separated populations and led to known morphological and genetic divergence. The DDM_today corresponds well with known contemporary wildcat distribution records, except where wildcats would be expected to be excluded (e.g. high human population densities, potential competitors, inaccessible islands). The DDM_today also corresponds closely with the results of recent studies on skull morphometrics and phylogeography, which support hypothesized colonizations of Africa and Asia from Europe during the late Pleistocene. Although DDM palaeo-distributions are more uncertain, they correspond to expected dramatic declines in northern Eurasia during the LGM, and significant distributional decline in central Asia, the Sahara and southern Africa, owing to increased aridity during climate cooling. From the DDM₉₀₀₀ model moderate global warming is hypothesized to impact minimally on wildcats, except in the Middle East and south-west Asia.     

Copulation, the dynamics of sperm transfer and female refractoriness in the leafhopper Balclutha incisa (Hemiptera: Cicadellidae: Deltocephalinae)

Abstract.  Mature sperm of the leafhopper Balclutha incisa (Matsumara) (Cicadellidae: Auchenorrhyncha: Hemiptera) are stored as a series of sperm bundles within seminal vesicles prior to ejaculation. During transfer, sperm are pumped from the vesicles into the ejaculatory duct to the complex aedeagus. Sperm transfer is marked by a c . 30-fold expansion of the spermatheca to accommodate both sperm and seminal fluid. Sperm number increases exponentially with male age, reaching a maximum of 700 000 after 14 days, while the number of sperm available on days 2–5 is between 70 000 and 100 000. During mating, maximum sperm transfer occurs after 7 min and mating is complete after about 10 min. Ejaculate size, defined by both sperm and associated accessory gland fluid, is influenced by male mating status and the interval since the previous mating. There is a positive correlation between duration of copulation and both ejaculate and the time to subsequent mating. Sperm are more likely to be retained in the testes during mating by males of 2–5 days post-emergence than older males. The number of sperm received by the female can be manipulated experimentally by mating males once (medium ejaculate) or twice (small ejaculate) immediately after their first mating. Females that receive small ejaculates from sperm-depleted males have a far shorter refractory period than females receiving medium to large ejaculates. Both ejaculate size and the time after males have mated influence the female post-mating refractory period as measured by the female's responsiveness to male sexual signalling.     

Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190 000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R₁ lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14 000–16 000 lines would be sufficient for 90% gene tagging coverage in M. truncatula . This is in contrast to more than 500 000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula , and could be a primary choice for other plant species with large genomes.     

Patterns of gene flow in two species of eel-tailed catfish, Neosilurus hyrtlii and Porochilus argenteus (Siluriformes: Plotosidae), in western Queensland's dryland rivers

Using genetic techniques (mtDNA, microsatellites and allozymes), this study investigated patterns of gene flow in two plotosid catfish species ( Neosilurus hyrtlii and Porochilus argenteus ), at various hierarchical scales in the dryland rivers of western Queensland, Australia. The study area constituted two major catchments, Cooper and Darling, representing arid and semiarid systems, respectively. Results generally conformed to expectations, with high levels of gene flow observed within catchments and limited contemporary gene flow evident across catchment boundaries. However, the isolation between catchments was more recent than expected, occurring approximately 40 000–72 000 years ago. Also contrary to predictions, genetic structure within the Cooper catchment did not fit the stream hierarchy model of genetic differentiation, which there was evidence of in the Darling catchment. This was hypothesized to relate to the different climatic regimes and hydrological inputs in each system, leading to a more genetically homogeneous system in the Cooper than in the Darling system.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 457–467.     

2-Methylhopanoids are maximally produced in akinetes of Nostoc punctiforme: geobiological implications

2-Methylhopanes, molecular fossils of 2-methylbacteriohopanepolyol (2-MeBHP) lipids, have been proposed as biomarkers for cyanobacteria, and by extension, oxygenic photosynthesis. However, the robustness of this interpretation is unclear, as 2-methylhopanoids occur in organisms besides cyanobacteria and their physiological functions are unknown. As a first step toward understanding the role of 2-MeBHP in cyanobacteria, we examined the expression and intercellular localization of hopanoids in the three cell types of Nostoc punctiforme : vegetative cells, akinetes, and heterocysts. Cultures in which N. punctiforme had differentiated into akinetes contained approximately 10-fold higher concentrations of 2-methylhopanoids than did cultures that contained only vegetative cells. In contrast, 2-methylhopanoids were only present at very low concentrations in heterocysts. Hopanoid production initially increased threefold in cells starved of nitrogen but returned to levels consistent with vegetative cells within 2 weeks. Vegetative and akinete cell types were separated into cytoplasmic, thylakoid, and outer membrane fractions; the increase in hopanoid expression observed in akinetes was due to a 34-fold enrichment of hopanoid content in their outer membrane relative to vegetative cells. Akinetes formed in response either to low light or phosphorus limitation, exhibited the same 2-methylhopanoid localization and concentration, demonstrating that 2-methylhopanoids are associated with the akinete cell type per se . Because akinetes are resting cells that are not photosynthetically active, 2-methylhopanoids cannot be functionally linked to oxygenic photosynthesis in N.   punctiforme .     

Parasitism by the mite Trombidium breei on four U.K. butterfly species

Abstract 1.  The incidence of parasitism by larvae of the mite species Trombidium breei was reported in one population of the lycaenid butterfly Polyommatus icarus , four populations of the satyrine butterfly Maniola jurtina , one population of the satyrine butterfly Aphantopus hyperanthus , and two populations of the satyrine butterfly Pyronia tithonus , as well as on one specimen of the dipteran Alophorus hemiptera . A considerable proportion of butterflies (11-50%) was infested in all study populations.
2. The pattern of infestation was examined in detail in M. jurtina . Males had a significantly higher incidence of infestation than females, and middle-aged butterflies had a higher incidence of infestation than old or young butterflies. The incidence of infestation peaked in the middle of the flight season, and this seasonal effect was independent of the effect of butterfly age.
3. Using a model based on capture-recapture data, it was estimated that a hypothetical ideal male M. jurtina that lives exactly the mean expected lifespan of 9-10 days has an approximately 75% chance of becoming infested with mites at least once during its lifetime, a mean time to first infestation of 3-4 days, and an average infestation persistence time of 2-3 days.
4. Capture-recapture data failed to show any effect of mite infestation on the lifespan or within-habitat movement rate of M. jurtina .
5. In experiments in which individual butterflies were taken out of their normal habitat and released, M. jurtina and P. tithonus that were infested with mite larvae did not differ from uninfested individuals in the efficiency with which they returned to suitable habitat. Thus, parasitism by T. breei larvae had no detectable effects on flight performance or orientation ability.
6. The results suggest that trombidiid mite larvae have limited potential in the biological control of insect pests.     

Relative influences of current and historical factors on mammal and bird diversity patterns in deglaciated North America

Aim To investigate the relative contributions of current vs. historical factors in explaining broad‐scale diversity gradients using a combination of contemporary factors and a quantitative estimate of the temporal accessibility of areas for recolonization created by glacial retreat following the most recent Ice Age. Location The part of the Nearctic region of North America that was covered by ice sheets during the glacial maximum 20 000 BP. Methods We used range maps to estimate the species richness of mammals and terrestrial birds in 48 400 km² cells. Current conditions in each cell were quantified using seven climatic and topographical variables. Historical conditions were estimated using the number of years before present when an area became exposed as the ice sheets retreated during the post‐Pleistocene climate warming. We attempted to tease apart contemporary and historical effects using multiple regression, partial regression and spatial autocorrelation analysis. Results A measure of current energy inputs, potential evapotranspiration, explained 76–82% of the variance in species richness, but time since deglaciation explained an additional 8–13% of the variance, primarily due to effects operating at large spatial scales. Because of spatial covariation between the historical climates influencing the melting of the ice sheet and current climates, it was not possible to partition their effects fully, but of the independent effects that could be identified, current climate explained two to seven times more variance in richness patterns than age. Main Conclusions Factors acting in the present appear to have the strongest influence on the diversity gradient, but an historical signal persisting at least 13 000 years is still detectable. This has implications for modelling changes in diversity patterns in response to future global warming.     

Contribution of Genome-Wide Association Studies to Scientific Research: A Bibliometric Survey of the Citation Impacts of GWAS and Candidate Gene Studies Published during the Same Period and in the Same Journals

In genetic epidemiology, genome-wide association studies (GWAS) are used to rapidly scan a large set of genetic variants and thus to identify associations with a particular trait or disease. The GWAS philosophy is different to that of conventional candidate-gene-based approaches, which directly test the effects of genetic variants of potentially contributory genes in an association study. One controversial question is whether GWAS provide relevant scientific outcomes by comparison with candidate-gene studies. We thus performed a bibliometric study using two citation metrics to assess whether the GWAS have contributed a capital gain in knowledge discovery by comparison with candidate-gene approaches. We selected GWAS published between 2005 and 2009 and matched them with candidate-gene studies on the same topic and published in the same period of time. We observed that the GWAS papers have received, on average, 30±55 citations more than the candidate gene papers, 1 year after their publication date, and 39±58 citations more 2 years after their publication date. The GWAS papers were, on average, 2.8±2.4 and 2.9±2.4 times more cited than expected, 1 and 2 years after their publication date; whereas the candidate gene papers were 1.5±1.2 and 1.5±1.4 times more cited than expected. While the evaluation of the contribution to scientific research through citation metrics may be challenged, it cannot be denied that GWAS are great hypothesis generators, and are a powerful complement to candidate gene studies.     

Spiegelzymes? Mirror-Image Hammerhead Ribozymes and Mirror-Image DNAzymes,an Alternative to siRNAs and microRNAs to Cleave mRNAs In Vivo?

With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes®) which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers) discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids), and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA) in these models can show either right- or left-handedness. This is a very new observation, suggesting that molecular symmetry of enantiomeric nucleic acids is broken down.     

ATTRACT-EM: A New Method for the Computational Assembly of Large Molecular Machines Using Cryo-EM Maps

Many of the most important functions in the cell are carried out by proteins organized in large molecular machines. Cryo-electron microscopy (cryo-EM) is increasingly being used to obtain low resolution density maps of these large assemblies. A new method, ATTRACT-EM, for the computational assembly of molecular assemblies from their components has been developed. Based on concepts from the protein-protein docking field, it utilizes cryo-EM density maps to assemble molecular subunits at near atomic detail, starting from millions of initial subunit configurations. The search efficiency was further enhanced by recombining partial solutions, the inclusion of symmetry information, and refinement using a molecular force field. The approach was tested on the GroES-GroEL system, using an experimental cryo-EM map at 23.5 Å resolution, and on several smaller complexes. Inclusion of experimental information on the symmetry of the systems and the application of a new gradient vector matching algorithm allowed the efficient identification of docked assemblies in close agreement with experiment. Application to the GroES-GroEL complex resulted in a top ranked model with a deviation of 4.6 Å (and a 2.8 Å model within the top 10) from the GroES-GroEL crystal structure, a significant improvement over existing methods.     

The effect of a seasonal time constraint on development time, body size, condition, and morph determination in the horned beetle Allomyrina dichotoma L. (Coleoptera: Scarabaeidae)

Abstract.  1. In horned beetles selection favours males that adjust their investment in horn development in relation to cues that predict adult body size. Here it is shown that in the Japanese horned beetle, Allomyrina dichotoma . There is a significant discontinuity in the horn length body size allometry. This can be described as a linear relationship that is shifted towards an increased horn length to body length ratio in males with horns longer than 16 mm.
2. Larval nutrition explains morph determination in A. dichotoma . However, unlike other species, variation in larval nutrition was the result of a seasonal time constraint that limits the time available for feeding prior to the onset of winter diapause.
3. Even when eggs were reared with an ad libitum food supply, minor morphs were still observed. Individuals that were oviposited later in the season had less time to feed, shorter development times, eclosed as smaller individuals and, in the case of males, were more likely to be hornless. Major morphs, minor morphs, and females all reduced their body size in response to seasonal time constraints in the same way. However, males that were laid later in the season had faster development times than females laid at the same time, but showed no reduction in their size relative to females, suggesting seasonal time constraints increase growth rates in males but not in females.
4. No evidence was found that seasonal time constraints resulted in a reduction of size-corrected fat reserves at eclosion, or that minor morphs gained any developmental advantage by reducing investment in horn length.     

Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli.

Crude extracts from Escherichia coli were screened for any broad-specificity endoribonuclease after the cell proteins were fractionated by size. In a mutant lacking the gene for RNase I (molecular mass, 27,156 Da), the only such activities were also in the size range of 23 to 28 kDa. Fractionation by chromatography on a strong cation-exchange resin revealed only two activities. One of them eluted at a salt concentration expected for RNase M and had the specificity of RNase M. It preferred pyrimidine-adenosine bonds, could not degrade purine homopolymers, and had a molecular mass of approximately 27 kDa (V. J. Cannistraro and D. Kennell, Eur. J. Biochem. 181:363-370, 1989). A second fraction, eluting at a higher salt concentration, was active against any phosphodiester bond but was about 100 times less active than are RNase I and RNase I* (a form of RNase I) in the wild-type cell. On the basis of sizing-gel chromatography, this enzyme had a molecular mass of approximately 24 kDa. We call it RNase R (for residual). RNase R is not an abnormal product of the mutant rna gene; a cell carrying many copies of that gene on a plasmid did not synthesize more RNase R. Our search for broad-specificity endoribonucleases was prompted by the expectation that the primary activities for mRNA degradation are expressed by a relatively small number of broad-specificity RNases. If correct, the results suggest that the endoribonucleases for this major metabolic activity reside in the 24- to 28-kDa size range. Endoribonucleases with much greater specificity must have as primary functions the processing of specific RNA molecules at a very limited number of sites as steps in their biosynthesis. In exceptional cases, these endoribonucleases inactivate a specific message that has such a site, and they can also effect total mRNA metabolism indirectly by a global disturbance of the cell physiology. It is suggested that a distinction be made between these processing and degradative activities.     

Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp -fusion gene expression and for protein depletion experiments in S. pneumoniae . Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.     

Seven-Pass Transmembrane Cadherins: Roles and Emerging Mechanisms in Axonal and Dendritic Patterning

The Flamingo/Celsr seven-transmembrane cadherins represent a conserved subgroup of the cadherin superfamily involved in multiple aspects of development. In the developing nervous system, Fmi/Celsr control axonal blueprint and dendritic morphogenesis from invertebrates to mammals. As expected from their molecular structure, seven-transmembrane cadherins can induce cell–cell homophilic interactions but also intracellular signaling. Fmi/Celsr is known to regulate planar cell polarity (PCP) through interactions with PCP proteins. In the nervous system, Fmi/Celsr can function in collaboration with or independently of other PCP genes. Here, we focus on recent studies which show that seven-transmembrane cadherins use distinct molecular mechanisms to achieve diverse functions in the development of the nervous system.