Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis.

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

Parenting, Effortful Control, and Adolescents' Externalizing Problem Behavior: Moderation by Dopaminergic Genes.

Research shows that genetics and effortful control play an important role in the link between parenting and problem behavior. However, little is known about how these factors act simultaneously. This article used a moderated mediation model to examine whether effortful control mediated the link between parenting and externalizing problem behavior, and whether dopaminergic genes (i.e., polygenic index score including DAT1, DRD2, DRD4, COMT) moderated this link. Two three-wave studies were conducted on community samples (adolescents: Study 1: N = 457; Mage = 15.74; Study 2: N = 221; Mage = 12.84). There was no mediation by effortful control, but a moderation by dopaminergic reactivity was observed. Despite inconsistent evidence, this article indicates that the development of externalizing problem behavior is subject to genetic characteristics and parenting.

Toe modifications in hind feet shoes optimise hoof-unrollment in sound Warmblood horses at trot.

REASONS FOR PERFORMING STUDY: It was shown that rolled-toe shoes smooth hoof-unrollment and thereby reduce peak loading of forelimbs in trotting Warmblood horses. Shoe design and shoeing technique for hind feet have been modified over recent decades from fully fitted, toe-clipped shoes to set-back, side-clipped shoes. OBJECTIVES: To study the effects of different shoe modifications on loading and movement of the hind hoof. METHODS: Ten clinically sound Warmblood horses were shod with 3 types of hind shoe: first, fully fitted, toe-clipped shoes, followed randomly by side-clipped shoes without a rolled toe that were set back by half the thickness of the hoof wall in the plantar direction and identical side-clipped shoes with a rolled toe, with 2 days between shoeing sessions to adapt to the shoes. Then horses were trotted in a straight line over a pressure-force plate combination. Hoof dynamics were compared statistically with the fully fitted shoe condition using a general linear model repeated measures test (P<0.05). RESULTS: There were no significant differences in the characteristics of limb timing, such as stance time and breakover duration nor the peak vertical and horizontal ground reaction force. By setting back the hind shoes, the centre of pressure at toe-off was positioned less dorsally. The rolled toe resulted in a smoother shift of the centre of pressure and thus more fluent hoof-unrollment. Both alternative types of shoe allowed a more lateral orientation of the centre of pressure at toe-off. CONCLUSIONS AND POTENTIAL RELEVANCE: Toe modifications of hind shoes can influence hindlimb kinematics positively. Setting the hind shoe back and rolling the toe leads to smoother hoof-unrollment, which enables the horse to coordinate movement correctly without loss of propulsion. Thus, shoe modifications might facilitate movement and thereby help prevent overload injuries.

Lateralised motor behaviour leads to increased unevenness in front feet and asymmetry in athletic performance in young mature Warmblood horses.

REASON FOR PERFORMING STUDY: Foot stance in grazing significantly influences hoof conformation and development from foal to yearling age. OBJECTIVES: To conduct a longitudinal study to establish if the relationship between motor laterality and uneven front feet persisted in 3-year-old horses at the time of studbook selection and to investigate if such laterality and unevenness might influence the horses' ability to perform symmetrically while trotting, cantering and free jumping. METHODS: Seventeen clinically sound but untrained (with only minimal experience of handling) and sound Warmblood horses that had participated in a previous study were assessed as per the protocol reported. Laterality was tested in a preference test (PT) and z-values were calculated for analysis purposes. Laterality and hoof unevenness were related to both relative limb length and relative head size, while the ability to perform symmetrically was tested in free trot-canter transitions and free jumping exercises. Differences in performance between horses with and without a limb preference in the PT and those with 'uneven' and 'even' feet were tested for differences in performance metrics using Students' t test, while linearity was tested using a regression analysis (P<0.05). RESULTS: Significant laterality was still present in 24% of the 3-year-old horses and the relationship between laterality and uneven feet pairs was stronger than at foal and yearling stages. Horses with significant motor laterality had almost 4 times more unevenness, a smaller head and longer limbs and the relationship between body conformation and laterality was still present. There was a strong linear relation between unevenness, laterality and a bias or side preference for trot-canter transitions. However, this relationship was not significant during the free jumping exercise. CONCLUSION: Motor laterality and uneven feet pairs were still present and significantly related in the 3-year-old horses and both variables were also strongly related to sidedness in trot-canter transitions. POTENTIAL RELEVANCE: Warmblood studbooks should include quantitative data on laterality at the time of studbook admission as part of the selection criteria.

Spectral diffusion and electron-phonon coupling of the B800 BChl a molecules in LH2 complexes from three different species of purple bacteria.

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

The use of force plate measurements to titrate the dosage of a new COX-2 inhibitor in lame horses.

REASONS FOR PERFORMING STUDY: Lameness is a highly prevalent condition in horses and the principal cause of removal from athletic activity. In clinical studies to evaluate nonsteroidal anti-inflammatory drug therapies, force plates are commonly used to assess improvement of lameness objectively. HYPOTHESIS: To use a force plate to determine the optimal dose of a new COX-2 inhibitor (firocoxib) that will reduce lameness, when administered orally to horses once daily. METHODS: Sixty-four horses that exhibited chronic lameness presumed due to osteoarthritis, including navicular disease, in at least one of the frontlimbs and at a stable level of severity, were included. Horses were treated per os s.i.d. for 7 days as follows: vehicle control, firocoxib at 0.05, 0.1 or 0.25 mg/kg bwt. Force plate analysis of each horse was done for the selected (most) lame frontlimb at trot. Once between Days -19 and -4 (initial examination), and again on Day -2 or -1 (baseline), pretreatment force plate assessments were performed, and thereafter horses were assessed on Days 0, 2 and 6, approximately 10 h post treatment each time. Peak vertical force (PVF) and lameness grades at initial examination and at baseline, and their change from baseline in the 4 different treatment groups were analysed statistically at a significance level of P < 0.05. RESULTS: The PVF results were found to be superior to vehicle control already at Day 0 for 0.25 mg/kg bwt and at Days 2 and 6 for 0.1 and 0.25 mg/kg bwt (P < 0.05). Mean clinical lameness for both concentrations decreased > 1 grade at Day 6. CONCLUSIONS AND CLINICAL RELEVANCE: With the dosage of 0.25 mg/kg bwt lameness did not improve more than with 0.1 mg/kg bwt. Thus, 0.1 mg/kg bwt s.i.d. was considered to be the effective dose at reducing chronic lameness in horses presumed due to osteoarthritis, including navicular disease.

Shoeing sound warmblood horses with a rolled toe optimises hoof-unrollment and lowers peak loading during breakover.

REASONS FOR PERFORMING STUDY: Overload injuries in sport horses commonly occur; shoeing techniques are believed to be important in prevention of these injuries, but there is a paucity of scientific information identifying the potential connection. OBJECTIVES: To test a horseshoe with a modified rolled toe designed to ease the process of breakover and decrease loading of lesion-prone structures of the distal limb. METHODS: Twenty clinically sound Warmblood horses trotted over a track containing a pressure/force measuring system and 6 infrared cameras. The horses were measured with 2 types of shoes, standard flat shoes and shoes with a rolled toe. The shoeing procedure was randomised and horses had 2 days between measurements to adapt to the shoes. RESULTS: Limb placement and timing characteristics, e.g. breakover duration, did not change significantly. There was an improvement in the ease of movement to roll over the toe in the shoes with a rolled toe, due mainly to a smoother hoof-unrollment pattern. The peak indicative moment decreased substantially at the onset of breakover in the shoe with the rolled toe. CONCLUSIONS: With a rolled toe the process of hoof-unrollment is smoother, which improves the coordination of this process, and lowers peak loading of the distal limb during breakover. POTENTIAL RELEVANCE: This study stresses the importance of proper shoeing in sound horses, showing that shoe modifications can optimise the loading characteristics of the distal limb and therefore might be a means to prevent sport horses from overload injuries.

Hoof growth between two shoeing sessions leads to a substantial increase of the moment about the distal, but not the proximal, interphalangeal joint.

REASONS FOR PERFORMING STUDY: There is little insight into the effects of routine farriery on the internal structures of the distal limb in sound horses. OBJECTIVES: To measure the effect of change in hoof conformation during a shoeing interval on the moments about the proximal and distal interphalangeal joints (PIPJ, DIPJ) and to determine whether and how the horse compensates for this change in hoof conformation. METHODS: Both front feet of 9 sound Warmblood horses were measured while standing on a pressure-force measuring system and radiographed in a lateromedial direction shortly after shoeing and 8 weeks later. From these data, ground reaction forces (GRF) and lever arms were measured in order to calculate joint moments. RESULTS: After 8 weeks, the moment about the PIPJ did not increase significantly, but the moment about the DIPJ did so, indicating a compensatory mechanism for a change in hoof conformation in the DIPJ. CONCLUSIONS: Standing horses compensate for hoof conformation change during an 8-week shoeing interval, which leads to increased DIPJ extension and consequently an increased loading of the deep digital flexor tendon. POTENTIAL RELEVANCE: This study quantifies the effect of a shoeing interval on the internal structures of the foot and helps to determine an appropriate shoeing interval for individual horses in which the hoof with the lowest hoof angle is the best indicator. The exact determination of an optimal individual shoeing interval requires further study.

Uneven feet in a foal may develop as a consequence of lateral grazing behaviour induced by conformational traits.

REASONS FOR PERFORMING STUDY: Conformational traits are important in breeding, since they may be indicative for performance ability and susceptibility to injuries. OBJECTIVES: To study whether certain desired conformational traits of foals are related to lateralised behaviour while foraging and to the development of uneven feet. METHODS: Twenty-four Warmblood foals, born and raised at the same location, were studied for a year. Foraging behaviour was observed by means of weekly 10 min scan-sampling for 8 h. A preference test (PT) was developed to serve as a standardised tool to determine laterality. The foals were evaluated at age 3, 15, 27 and 55 weeks. The PT and distal limb conformation were used to study the relation between overall body conformation, laterality and the development of uneven feet. Pressure measurements were used to determine the loading patterns under the feet. RESULTS: About 50% of the foals developed a significant preference to protract the same limb systematically while grazing, which resulted in uneven feet and subsequently uneven loading patterns. Foals with relatively long limbs and small heads were predisposed to develop laterality and, consequently unevenness. CONCLUSIONS: Conformational traits may stimulate the development of laterality and therefore indirectly cause uneven feet.

Developmental aspects of distal limb conformation in the horse: the potential consequences of uneven feet in foals.

REASONS FOR PERFORMING STUDY: Distal limb conformation is generally accepted to be an important item with respect to performance and soundness in mature horses, but little is known about the developmental aspects. OBJECTIVES: To gain insight into the development of distal limb conformation and to assess the possible consequences of uneven feet in foals. METHODS: Conformation of the distal front limbs of 23 Warmblood foals was scored visually and measured using radiographs, at ages 27 and 55 weeks. At the same ages, pressure measurements were made under both front feet. RESULTS: At both ages the hoof-pastern axis was broken-backwards on radiographs, but only occasionally recognised as such, when scored by eye. Over time, the hoof angle decreased, while both the angles of the dorsal and solar surfaces of the distal phalanx (P3) increased and the parallelism between hoof wall and P3 improved. The foals with uneven feet at age 27 weeks showed a significant difference in distal limb loading that persisted until age 55 weeks. CONCLUSIONS: The alignment of the distal limb in the sagittal plane increased in a 6 month period. Visual assessment was not sensitive enough to appreciate this. The growth processes in the distal limb could not compensate for existing unevenness and ensuing asymmetrical limb loading. POTENTIAL RELEVANCE: Foals have a different conformation of the distal limb from mature horses, which should be taken into account when interpreting radiographs. Unevenness of the feet resulted in asymmetrical loading of the proximal and distal interphalangeal joint, which might lead to increased susceptibility to overload injuries and decreased performance at mature age.

Changes in location of centre of pressure and hoof-unrollment pattern in relation to an 8-week shoeing interval in the horse.

REASONS FOR PERFORMING STUDY: In order to optimise shoeing and shoeing conditions, it is essential to know how the horse adapts to a common shoeing interval. OBJECTIVES: To measure changes in location of the centre of pressure (CoP) and hoof-unrollment pattern during a shoeing interval and to assess whether these changes in CoP result directly from changes in hoof conformation or are also influenced by compensatory mechanisms. METHODS: Eighteen horses were trotted over a pressure-force measurement system shortly after shoeing and 8 weeks later. The position of the CoP was measured and also predicted using calculations based on changes in hoof conformation. RESULTS: The measured shift in CoP was less than calculated. This difference was largest in the hind feet. The hoof-unrollment pattern stayed basically the same in the front feet, but in the hind feet there was a substantial lateral shift of the trajectory of the CoP. CONCLUSIONS: Horses can compensate, to a certain extent, for changes in hoof conformation that develop during 8 weeks on shoes. As the capacity to compensate is less in the forelimbs, the relative increase in loading of these limbs during a shoeing interval is larger than in the hindlimbs. POTENTIAL RELEVANCE: This study provides essential basic data for the development of science-based shoeing techniques.

TYSON: robust searching, sorting, and selecting of single particles in electron micrographs.

We here present TYSON, a new program for automatic and semi-automatic particle selection from electron micrographs. TYSON employs a three-step strategy of searching, sorting and selecting single particles. In the first step, TYSON finds the positions of potential particles by one of three different methods: local averaging, template matching or local variance. The practical merits and drawbacks of these methods are discussed. In the second step, these potential particles are automatically sorted according to their probability of being true positives. Many criteria are provided for this sort. In the final -interactive- step, whole categories of poorly fitting false positives can be removed with a single mouse-click. We present results obtained using cryo-EM micrographs of both spherical virus particles and asymmetric particles. The procedures are fast and use of TYSON allowed, for example, some 20,000 particles to be selected in a single working day.

Dynamic pressure measurements for the detailed study of hoof balance: the effect of trimming.

REASONS FOR PERFORMING STUDY: Studies on hoof balance have, so far, only assessed the effects of strong and relatively unrealistic interventions due to technical limitations of measuring equipment. For the assessment of more subtle interventions, other techniques are necessary. OBJECTIVES: To test a sensitive pressure measurement system during locomotion and to set a standard for further studies by using the system to evaluate the effects of trimming. METHODS: Eighteen horses were measured before and after trimming with an interval of 4 weeks. Trimming was standardised to a straight hoof-pastern axis. The horses trotted over a pressure/force measuring system, with a temporal resolution of 240 Hz and a spatial resolution of 0.39 cm2. RESULTS: The preferred way of landing was lateral, asymmetrical in both front and hind feet. The duration of landing was shorter in forelimbs than in hindlimbs with an equal decreasing trimming effect. Horses had a fixed hoof-unrollment pattern; the centre of pressure (CoP) travelled towards a maximum lateral deviation and returned towards the dorsopalmar/plantar axis of the hoof. Trimming decreased the intra-individual left/right difference in maximum lateral displacement. CONCLUSIONS: The technique used provided easily accurate data to quantify hoof balance characteristics and to measure short-term trimming effects. POTENTIAL RELEVANCE: Determination of CoP patterns has added value in gait analysis and can improve our understanding of the effects of different interventions on hoof balance.

Structural organisation of the head-to-tail interface of a bacterial virus.

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-naïve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.

Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui.

Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where significant length variations between different species are known to occur. At each of these positions, with the exception of h45, there are also significant differences between the 23 S rRNAs of E. coli and Haloarcula marismortui. Plasmids carrying the insertions were introduced into an E. coli strain lacking all seven rrn operons. In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal. The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements. The results were compared both with the recently determined atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E. coli ribosomes. The insertion element in h45 was located by cryo-EM at a position corresponding precisely to that of the equivalent helix in H. marismortui. The insertion in h98 (which is entirely absent in H. marismortui) was similarly located at a position corresponding precisely to that predicted from the E. coli modelling studies. In the region of h9, the difference between the E. coli and H. marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure. In the case of h25 (which is about 50 nucleotides longer in H. marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed.

Escherichia coli RNA polymerase core and holoenzyme structures.

Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 A structure of the core RNA polymerase and a 9.5 A structure of the sigma(70) holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in ss'. All common RNA polymerase subunits (alpha(2), ss, ss') could be localized in both structures, thus suggesting the position of sigma(70) in the holoenzyme.

Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids.

Many annelids, including the earthworm Lumbricus terrestris, have giant cooperative respiratory proteins (molecular masses greater than 3.5 million Da) freely dissolved in the blood, rather than packaged in cells. These complexes, termed either erythrocruorins or hemoglobins, are assembled from many copies of both hemoglobin subunits and nonhemoglobin or "linker" subunits. In this paper, we present the crystal structure of Lumbricus erythrocruorin at 5.5-A resolution, which reveals a remarkable hierarchical organization of 144 oxygen-binding hemoglobin subunits and 36 nonhemoglobin linker subunits. The hemoglobin chains arrange in novel dodecameric substructures. Twelve trimeric linker complexes project triple-stranded helical coiled-coil "spokes" toward the center of the complex; interdigitation of these spokes appears crucial for stabilization. The resulting complex of linker chains forms a scaffold on which twelve hemoglobin dodecamers assemble. This structure specifies the unique, self-limited assemblage of a highly cooperative single molecule.

Tetramerisation of alpha-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca(2+)-dependent vesicular exocytosis from synaptosomes.

A novel procedure of alpha-latrotoxin (alpha LTX) purification has been developed. Pure alpha LTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca(2+), Mg(2+) or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified alpha LTX loses the ability to tetramerise spontaneously; the addition of Mg(2+) or Ca(2+) restores this ability. This suggests that alphaLTX has some intrinsically bound divalent cation(s) that normally support its tetramerisation. Single-particle cryoelectron microscopy and statistical image analysis have shown that: 1) the toxin has a non-compact, branching structure; 2) the alpha LTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 A-diameter channel in the centre. Both alpha LTX oligomers bind to the same receptors in synaptosomes and rat brain sections. To study the effects of the dimers and tetramers on norepinephrine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a preparation correlates with alpha LTX pore formation, suggesting that the tetramers are the pore-forming species of alpha LTX. The toxin actions mediated by the pore include: 1) Ca(2+) entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca(2+). The Ca(2+)-dependent alpha LTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca(2+) stores and functional phospholipase C (PLC). The Ca(2+)-dependent effect of the toxin is stronger when dimeric alpha LTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca(2+)-independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations.