Redefining dominance calculation: Increased competition flattens the dominance hierarchy in dairy cows.

Dominance hierarchies are known for mitigating conflicts and guiding priority of access to limited resources in gregarious animals. The dominance hierarchy of dairy cows is typically investigated using agonistic interactions, often monitored at the feed bunk right after fresh feed delivery when competition is high, resulting in frequent interactions. Yet, the outcome of agonistic interactions during times of high competition may be more influenced by cows' high valuation of fresh feed than their intrinsic attributes, such that the dominance hierarchy constructed using agonistic interactions under high versus low competition times might differ. We tested how the structure of the dominance hierarchy changes in relation to different levels of competition in a dynamic group of 48 lactating dairy cows over 10 mo, with 6 cows exchanged every 16 d, for a total of 159 cows. Using a validated algorithm, we continuously detected the actor and reactor of replacement behaviors in 30 feed bins as cows competed for feed. We also calculated the percentage of occupied feed bins to characterize competition at the moment of each replacement. These data were combined to create hierarchies using Elo ratings, separately for 25 occupancy levels ranging from 13% to 100%. For each 1% rise in feeder occupancy, hierarchy steepness fell by 2.41 × 10-3 ± 9.71 × 10-5 (SE), and the percentage of dyads where both cows replaced each other rose by 0.13% ± 0.01%. At the highest feeder occupancy level in comparison to the lowest one, we observed 7.57% more dyads in which the dominant individual (those that won more interactions at the lowest feeder occupancy) started to lose proportionally more. The magnitude of decrease in the winning rate of the dominant individual in those dyads also got amplified by 1.06 × 10-3% ± 1.37 × 10-4% (SE) for each 1% increase in feeder occupancy. These findings illustrate how inferred hierarchies vary with competition, with high competition flattening the hierarchy due to increased success of subordinate animals. We suggest that during heightened competition, increased valuation of resources can affect competitive success more than the individual's intrinsic dominance attributes. We recommend against calculating dominance hierarchies based on agonistic interactions during periods of high competition alone, and more generally urge researchers to differentiate agonistic interactions based on context when constructing dominance hierarchies.

Invited review: Social dominance in dairy cattle: A critical review with guidelines for future research.

Cattle are gregarious animals able to form social relationships. Dominance is one of the most widely studied social behaviors of dairy cattle, especially cows confined indoors. However, much of the past dairy cattle research has used an unstandardized approach, differing in definitions and conceptual understanding of dominance, as well as their methods of data collection and dominance calculation. The first of the 3 aims of this review is to evaluate how dominance relates to the social behavior of housed dairy cows. Cows engage in agonistic interactions to establish and reinforce dominance relationships. An individual's dominance is influenced by intrinsic characteristics, such as personality, and extrinsic factors, including group composition. When competing for resources, agonistic interactions can also be influenced by individual motivational differences, such as hunger, which may diminish the role of dominance in regulating competition. Our second aim is to critically review methods used to assess dominance in cows. This includes discussions on the effect of time and location of data collection on measured values as well as the viability and limitations of some dominance calculation methods. We propose that different methodologies lend themselves to different types of research questions. For example, the use of data stream-based methods that consider the sequence of interactions are useful for estimating how dominance fluctuates with changing conditions and can be used in a dynamically changing group. In contrast, matrix-based methods that aggregate social interactions may be best for identifying the social position of individuals and understanding how social characteristics influence the attributes of a stable hierarchy. Our third aim is to discuss the future of dominance research. We use a flowchart to illustrate guidelines for a more standardized approach to measuring dominance in cattle. We also identify areas in need of further conceptual clarification, suggest practical applications of dominance when managing dairy cattle, and discuss some limitations of dominance research.

Dissecting the substrate recognition of 3-O-sulfotransferase for the biosynthesis of anticoagulant heparin.

Heparin is a polysaccharide-based natural product that is used clinically as an anticoagulant drug. Heparan sulfate 3-O-sulfotransferase (3-OST) is an enzyme that transfers a sulfo group to the 3-OH position of a glucosamine unit. 3-OST is present in multiple isoforms, and the polysaccharides modified by these different isoforms perform distinct biological functions. 3-OST isoform 1 (3-OST-1) is the key enzyme for the biosynthesis of anticoagulant heparin. Here, we report the crystal structure of the ternary complex of 3-OST-1, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Comparisons to previously determined structures of 3-OST-3 reveal unique binding modes used by the different isoforms of 3-OST for distinguishing the fine structures of saccharide substrates. Our data demonstrate that the saccharide substrates display distinct conformations when interacting with the different 3-OST isoforms. Site-directed mutagenesis data suggest that several key amino residues, including Lys259, Thr256, and Trp283 in 3-OST-3 and Arg268 in 3-OST-1, play important roles in substrate binding and specificity between isoforms. These results deepen our understanding of the biosynthetic mechanism of heparan sulfate and provide structural information for engineering enzymes for an enhanced biosynthetic approach to heparin production.

Effects of group size on agonistic interactions in dairy cows: a descriptive study.

Group-housed cattle may engage in agonistic interactions over resources such as feed, which can negatively affect aspects of welfare. Little is known about how contextual factors such as group size influence agonistic behaviour. We explored the frequency of agonistic interactions at the feeder when cattle were housed in different-sized groups. We also explored the consistency of the directionality of agonistic interactions in dyads and of the number of agonistic interactions initiated by individuals across the group sizes. Four replicates of 50 cows each were assessed in two group-size phases. In Phase 1, cows were kept in one group of 50. In Phase 2, these same cows were divided into five groups of 10, maintaining stocking density (i.e., ratio of animals to lying stalls and feed bunk spaces). We measured agonistic replacements (i.e., interactions that result in one cow leaving the feed bin and another taking her place) at an electronic feeder using a validated algorithm. We used these data from Phase 1 to calculate individual Elo-ratings (a type of dominance score). Cows were then categorised into five dominance categories based upon these ratings. To ensure a consistent Elo-rating distribution between phases, two cows from each dominance category were randomly assigned to each small group of 10 cows. The mean ± SE number of replacements per cow was similar regardless of whether the cows were housed in groups of 50 (34.1 ± 2.4) or 10 (31.1 ± 4.5), although the groups of 10 were more variable. Further, 81.6 ± 7.7% (mean ± SD) of dyads had the same directionality across group sizes (i.e., the same individual won the majority of interactions in the dyad) and individuals were moderately consistent in the number of replacements they initiated (intraclass correlation coefficient = 0.62 ± 0.11; mean ± SD). These results indicate that the relationship between group size and agonistic behaviour is complex; we discuss these challenges and suggest new avenues for further research.

Automated, longitudinal measures of drinking behavior provide insights into the social hierarchy in dairy cows.

Dairy cows compete for feed and water access on commercial farms. In this study we used EloSteepness to assess the summed Elo winning probabilities (i.e., dominance) of 87 cows housed in a dynamic group and compared the resulting social hierarchies based on their steepness (i.e., the average degree of differences in winning probability between adjacently ranked individuals in the group, ranging from 0 to 1). We identified a hierarchy at the drinker with a steepness of 0.55 ± 0.02 (SD), whereas the hierarchy detected at the feeder during the same time period was 0.45 ± 0.02, indicating smaller dominance differences among cows when competing for feed compared with competing for water. Individual cows' winning probabilities at the feeder and drinker were moderately correlated (rs = 0.55), and cows at the lower and upper ends of the hierarchy showed good agreement. We compared the drinker hierarchy between hot (i.e., temperature-humidity index [THI] ≥72) and normal (i.e., THI <72) periods. The hierarchy steepness was similar in both hot (0.54 ± 0.03) and normal conditions (0.56 ± 0.03), and there was a strong correlation in cows' individual winning probabilities across these periods (rs = 0.87). Cows with higher winning probability visited the drinker less frequently (hot: rs = -0.40, normal: rs = -0.33) but had a higher average daily water intake (hot: rs = 0.38, normal: rs = 0.37). We also found evidence that individual cows' drinking times differ depending on their winning probability; cows with lower winning probability shifted their drinking times to before or after the visit peak after milking. Automatically identifying cows with consistently high or low winning probabilities using drinkers may help inform grouping decisions and water provision on farms.

Automated monitoring of brush use in dairy cattle.

Access to brushes allows for natural scratching behaviors in cattle, especially in confined indoor settings. Cattle are motivated to use brushes, but brush use varies with multiple factors including social hierarchy and health. Brush use might serve an indicator of cow health or welfare, but practical application of these measures requires accurate and automated monitoring tools. This study describes a machine learning approach to monitor brush use by dairy cattle. We aimed to capture the daily brush use by integrating data on the rotation of a mechanical brush with data on cow identify derived from either 1) low-frequency radio frequency identification or 2) a computer vision system using fiducial markers. We found that the computer vision system outperformed the RFID system in accuracy, and that the machine learning algorithms enhanced the precision of the brush use estimates. This study presents the first description of a fiducial marker-based computer vision system for monitoring individual cattle behavior in a group setting; this approach could be applied to develop automated measures of other behaviors with the potential to better assess welfare and improve the care for farm animals.

The structural basis for partitioning of the XRCC1/DNA ligase III-α BRCT-mediated dimer complexes.

The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-α. For efficient ligation, ligase III-α is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-α BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

The Effect of Placement and Group Size on the Use of an Automated Brush by Groups of Lactating Dairy Cattle.

Mechanical brushes are often provided on dairy farms to facilitate grooming. However, current brush designs do not provide data on their use, and thus little is known about the effects of group size and placement of brushes within the pen. The objectives of this study were to automatically detect brush use in cow groups and to investigate the influence of (1) group size and the corresponding cow-to-brush ratio and (2) brush placement in relation to the lying stalls and the feeding and drinking areas. We measured brush use in groups of 60, 48, 36, and 24 cows, with the brush placed either in the alley adjacent to the feed bunk and water trough or in the back alley. Cows used the brush for longer when it was placed in the feed/water alley compared to when placed in the back alley. Average brush use per cow increased when cows were housed in smaller groups, but the brush was never in use more than 50% of the day, regardless of group size. We conclude that brush use increases when availability is increased and when the brush is placed closer to the feed and water.

Structure of human ADAM-8 catalytic domain complexed with batimastat.

The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme-inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded ß-sheet and a catalytic Zn(2+) ion. However, unique differences within the S1' binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.

A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs.

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the "core" PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

Der p 5 crystal structure provides insight into the group 5 dust mite allergens.

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of approximately 3000 A(3) that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

The effects of cow dominance on the use of a mechanical brush.

An animal's social position within a group can influence its ability to perform important behaviours like eating and resting, but little is known about how social position affects the ability to express what are arguably less important but still rewarding behaviors, such as grooming. We set out to assess if dominance measured at the feeder is associated with increased use of a mechanical brush. Over a 2-year period, 161 dry cows were enrolled in a dynamically changing group of 20 individuals with access to a mechanical brush. We determined dominance using agonistic behaviors at the feeder and retrospectively analyzed brush use for the 12 most, and 12 least dominant individuals during the week before calving. Cows that were more dominant at the feeder used the brush more, especially during peak feeding times. Agonistic interactions at the brush did not differ between dominants and subordinates and were not related to brushing duration. These findings indicate that social position, calculated using competition for feed, affects mechanical brush access such that subordinates use the brush less than dominant cows independent of competition or time of day.

Conformational dependence of 13C shielding and coupling constants for methionine methyl groups.

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of (3)J (CSCC), (3)J (CSCH), and the isotropic shielding, sigma(iso). Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-(13)C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, (3)J (CSCC)/(3)J (COCC) approximately 0.7. It is further demonstrated that the (3)J (CSCH) coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of delta(13)Cepsilon. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large approximately 100 degrees chi(3) values. The utility of the delta(13)Cepsilon as a basis for estimating the gauche/trans ratio for chi(3) is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.

A closed conformation for the Pol lambda catalytic cycle.

Pol lambda is a family X member believed to fill short gaps during DNA repair. Here we report crystal structures of Pol lambda representing three steps in filling a single-nucleotide gap. These structures indicate that, unlike other DNA polymerases, Pol lambda does not undergo large subdomain movements during catalysis, and they provide a clear characterization of the geometry and stereochemistry of the in-line nucleotidyl transfer reaction.

Evolution from DNA to RNA recognition by the bI3 LAGLIDADG maturase.

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 A from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.

Slow conformational dynamics at C2'-endo nucleotides in RNA.

RNA molecules undergo local conformational dynamics on timescales spanning picoseconds to minutes. Slower local motions have the greater potential to govern RNA folding, ligand recognition, and ribonucleoprotein assembly reactions but are difficult to detect in large RNAs with complex structures. RNA SHAPE chemistry employs acylation of the ribose 2'-hydroxyl position to measure local nucleotide flexibility in RNA and is well-characterized by a mechanism in which each nucleotide samples unreactive (closed) and reactive (open) states. We monitor RNA conformational dynamics over distinct time domains by varying the electrophilicity of the acylating reagent. Select C2'-endo nucleotides are nonreactive toward fast reagents but reactive toward slower SHAPE reagents in both model RNAs and in a large RNA with a tertiary fold. We conclude, first, that the C2'-endo conformation by itself does not govern SHAPE reactivity. However, some C2'-endo nucleotides undergo extraordinarily slow conformational changes, on the order of 10(-4) s(-1). Due to their distinctive local dynamics, C2'-endo nucleotides have the potential to function as rate-determining molecular switches and are likely to play central, currently unexplored, roles in RNA folding and function.

Role of the catalytic metal during polymerization by DNA polymerase lambda.

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.

Magnesium-induced assembly of a complete DNA polymerase catalytic complex.

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.

Structural insight into the DNA polymerase beta deoxyribose phosphate lyase mechanism.

A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase beta (Pol beta) in mammalian base excision repair (BER). Pol beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.

Structural insights into DNA polymerase beta deterrents for misincorporation support an induced-fit mechanism for fidelity.

DNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase beta with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity.