Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.DOI:http://dx.doi.org/10.7554/eLife.21007.001 Accurate chromosome segregation in eukaryotes requires the coordinated action of hundreds of proteins. Subsets of these assemble on centromeric chromatin that is epigenetically specified by the enrichment of centromeric protein A (CENP-A), a variant of Histone H3 . These aThe structural core of the kinetochore assembles from two large multi-subunit complexes, the CCAN (constitutive centromere associated network), and the KMN , which are respectively proximal and distal to the centromeric chromatin . The affWhereas CENP-C directly binds to CENP-A containing nucleosomes  and has Depletions of CENP-C or CENP-T, or prevention of Cyclin-dependent kinase (CDK) phosphorylation of CENP-T all affect proper recruitment of MIS12C and NDC80C to the kinetochore . StudyinThe CENP-T-dependent pathway, on the other hand, remains less well characterized. It is firmly established that CENP-T interacts directly with NDC80C, with (humans and chicken) or without (budding yeast) prior phosphorylation by CDK1:Cyclin B of sequence motifs in the CENP-T N-terminal region . ComplicThe ability of kinetochores to form multiple low-affinity linkages with microtubules is at the basis of popular models of kinetochore-microtubule attachment such as the Hill’s sleeve . It is aTo address this question, we have embarked on an effort of biochemical reconstitution of kinetochores. This recently allowed us to report that the CENP-A nucleosome recruits two copies of the CCAN complex . In thisbona fide binding sites from sites that do not interact.To obtain insight into the molecular mechanism of CENP-T mediated recruitment of the human outer kinetochore, we set out to reconstitute this process using purified components. Previous studies identified multiple phosphorylation sites in CENP-T and showed that its phosphorylation contributes to the recruitment of NDC80C . Confirm2-101 constructs in which three of the four CDK phosphorylation sites were mutated to Alanine. Constructs retaining either Thr11 or Thr85 bound to SPC24:SPC25 in a phosphorylation dependent manner, whereas constructs retaining either Thr27 or Ser47, or having all four sites mutated to Alanine, did not bind SPC24:SPC25  was in vitro phosphorylated by CDK1:Cyclin B and subsequently incubated with a threefold molar excess of SPC24:SPC25. CENP-T was part of a broad peak with two SPC24:SPC25-containing species with approximate retention volumes of 1.2 and 1.35 ml  fraction of the CENP-T:NDC80C mixture had a more heterogeneous appearance than NDC80C in isolation  and 4. W2-101, already at equimolar concentrations, indicative of higher binding affinity (unpublished data). In apparent contradiction with these observations, however, it has also been reported that the phosphorylation of human CENP-T on Thr195, within a weakly conserved CDK1 consensus motif  experiments. Both MIS12C and CENP-T sedimented with relatively high frictional ratios, indicative of an elongated shape or of lack of a defined conformation, respectively . To anal2-373 fusion proteins to an ectopically integrated lac operon (lacO) array in U2OS cells. As previously reported  CENP-TT11A or CENP-TT85A associate with MIS12C and NDC80C in 1:1:2 ratio; and 3) CENP-TT11A-T85A associates with MIS12C and NDC80C in 1:1:1 ratio. The behavior of the complete set of CENP-T mutants upon incubation with MIS12C and/or NDC80C is displayed in To test directly if MIS12C that is bound to phosphorylated CENP-T retains its ability to bind NDC80C, we returned to our reconstituted system and incubated different constructs of CENP-T with MIS12C and a molar excess of NDC80C. Phosphorylated CENP-T integrated in large complexes whose elution volume from the SEC column was smaller than that of MIS12:NDC80 complexes. Our set of CENP-T mutants enabled a systematic analysis of these CENP-T:MIS12C:NDC80C assemblies. In addition to recapitulating observations made for CENP-T:NDC80C  and CENP2 complexes  abolished the binding of MIS12C with CENP-C  and the lower MIS12C:CENP-T192-215 binding affinity (dK ~30 μM)  . Longer e manner . As expeg mutant  did not g mutant . These rFull assembly of the outer kinetochore is restricted to mitosis and is tightly regulated, but the molecular requirements for this regulation remain poorly understood. Here, we have reported a thorough dissection of the role of CENP-T and its phosphorylation by CDK1 in this process. Depletion of CENP-T almost completely abolishes the ultrastructure of the outer kinetochore  and phenT11 and CENP-TT85 and an entire KNL1C:MIS12C:NDC80C via phosphorylated CENP-TS201. Because the binding of CENP-C and CENP-T to MIS12C is competitive, these findings support a model in which CENP-C and CENP-T act in parallel to recruit two MIS12Cs, two KNL1Cs, and up to four NDC80Cs in a CDK-regulated manner  but only ~4 nm apart in the absence of tension (nocodazole treated cells), as determined by immunofluorescence and immuno-electron microscopy . Moreove3 complexes structurally rearrange in the presence of microtubules and if the presence of multiple NDC80Cs contributes to the stabilization of the kinetochore-microtubule interface observed in the presence of tension -Codon-plus-RIL cells containing the CENP-T encoding pGEX-6P-2rbs vector were grown at 37°C in Terrific Broth in the presence of Chloramphenicol and Ampicillin to an OD600 of ~0.8. Protein expression was induced by the addition of 0.35 mM IPTG and cells were incubated ~14 hr at 20°C. All following steps were performed on ice or at 4°C. Cell pellets were resuspended in buffer A  glycerol and 1 mM TCEP) supplemented with 0.5 mM PMSF, protease-inhibitor mix HP Plus (Serva) and DNaseI (Roche), lysed by sonication and cleared by centrifugation at 108,000g for 30 min. The cleared lysate was applied to a 5 ml GSTrap FF column  equilibrated in buffer A. The column was washed with approximately 50 column volumes of buffer A and bound protein was eluted in buffer A by cleavage of the GST-tag with PreScission protease for ~14 hr. The eluate was diluted 10-fold with buffer B  and applied to a 5 ml HiTrap Heparin HP column  pre-equilibrated in buffer B with 25 mM NaCl. Bound protein was eluted in buffer B using a linear gradient from 25 mM to 800 mM NaCl in 16 column volumes. Selected fractions were concentrated  and either used for Sortase-mediated labeling (see below) or directly applied to an equilibrated Superdex 200 10/300 column . SEC was performed with a 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM TCEP mobile phase at a flow rate of 0.4 ml/min, and the relevant fractions were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80°C.cDNAs encoding GST-CENP-T6xHis) were combined on a pFL (g for 30 min. The cleared lysate was filtered (0.8 μM) and applied to a 5 ml HisTrap FF  equilibrated in buffer A with 20 mM imidazole. The column was washed with approximately 50 column volumes of buffer A with 20 mM imidazole and bound proteins were eluted in buffer A with 300 mM imidazole. Relevant fractions were pooled, diluted 5-fold with buffer A with 25 mM NaCl and applied to a 6 ml ResourceQ column  equilibrated in the same buffer. Elution of bound protein was achieved by a linear gradient from 25 mM to 400 mM NaCl in 30 column volumes. Relevant fractions were concentrated in 30 kDa molecular mass cut-off Amicon concentrators (Millipore) in the presence of an additional 200 mM NaCl and applied to a Superdex 200 10/300 or a Superose 6 10/300 column  equilibrated in size-exclusion chromatography buffer . Size-exclusion chromatography was performed under isocratic conditions at the recommended flow rates and the relevant fraction were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80°C. Expression and purification of the full-length MIS12 complex  was identical but size-exclusion chromatography was performed in 50 mM Hepes, pH 8.0, 200 mM NaCl, 1 mM and no additional NaCl was added before the concentration of the relevant fractions of the ion-exchange chromatography step. MIS12CNano, designed for structural analysis  and combined in a pBIG1A vector  with N-tS. aureus Sortase  or fluorescein amidite (FAM) (Genscript). Labeling was performed for ~14 hr at 4°C in the presence of 10 mM CaCl2 using molar ratios of Sortase, CENP-T, and peptide of approximately 1:20:200. CENP-T was separated from Sortase and the unreacted peptides by size-exclusion chromatography as described above. Based on the absorbance at 280 nm and 495 nm (FAM) or 555 nm (TMR), a labeling efficiency of >90% was achieved.Purified  Sortase  or the S Sortase  were use2-101 fragments and CENP-T2-373 used in 2. Reaction mixtures were kept at 30°C for 30–60 min and then used in binding experiments.CENP-T2, 10% glycerol, 90 U/ml benzonase (Sigma) and 0.1% NP-40 supplemented with protease inhibitor cocktail (Serva) and PhosSTOP phosphatase inhibitors (Roche)]. Clarified extracts were pre-cleared with protein A–Sepharose  for 1 hr at 4°C before a 2 hr incubation with anti-CENP-T/W antibody (2-373 (in vitro samples) or the uniprot human database (in vivo samples) with deamidation (NQ), oxidation (M) and phosphorylation (STY) as variable modifications and carbamidomethylation (C) as fixed modification. A false discovery rate cut off of 1% was applied at the peptide and protein levels and as well on the site decoy fraction  mounted with a column  equilibrated in size-exclusion chromatography buffer  and operated at or near the recommended flow rate. Fractions of 50–100 μL were collected in 96-well plates and analyzed by SDS-PAGE. The absorbance of fluorescently labeled proteins was followed during chromatography using the ÄKTAmicro Monitor UV-900  and after SDS-PAGE using a ChemiDoc MP system (Bio-Rad).All samples were exchanged into fresh buffer . ITC measurements were performed at 20°C on an ITC200 microcalorimeter . In each titration, the protein in the cell (at a 10–30 μM concentration) was titrated with 25 × 4 μl injections  of protein ligand (at 10-fold higher molar concentration). The following synthetic peptides  were used Thr11 DST(p)PRTLLRRVLDTAYA, Thr85 EQT(p)PRTLLKNILLTAYA, Thr27 PRT(p)PRRPRSARAGARYA, and Thr47 TAS(p)PRKLSGQTRTIARYA. Injections were continued beyond saturation to allow for determination of heats of ligand dilution. Data were fitted by least-square procedure to a single-site binding model using ORIGIN software package .5-FAMCENP-T peptides or of a 5-FAMCENP-C1-21:MIS12C complex. These were mixed with increasing concentrations of the CENP-C1-71, CENP-T195-215, or MIS12C variants at the indicated concentrations in binding buffer . The reaction mixtures were allowed to equilibrate for approximately 15 min at room temperature. Fluorescein (5-FAM) was excited with polarized light at 470 nm, and the emitted light was detected at 525 nm through both horizontal and vertical polarizers. No change in the observed signal (or the underlying observed dissociation constant) was detected after one-hour incubation on ice. Polarization values are shown as mean ± standard error of the mean for three replicates. Dissociation constant values (Kd) were obtained by fitting the fluorescence polarization data by non-linear least square method to a single-site binding model using the Origin software.Fluorescence polarization measurements were performed with a Safire two instrument (Tecan) at 30°C using Corning 384 Well Low Volume Black Round Bottom Polystyrene NBS microplates. Reaction volumes of 20 μL contained a fixed concentration (20 nM) of one of the indicated c(s) distribution. The partial specific volumes of the proteins, buffer density and buffer viscosity were estimated using the program SEDNTERP. Data figures were generated using the program GUSSI.Sedimentation velocity AUC was performed at 42,000 rpm at 20°C in a Beckman XL-A ultracentrifuge. Purified protein samples were diluted to 15–30 μM in a buffer containing 20 mM Tris-HCl, 150 mM NaCl and 1 mM TCEP and loaded into standard double-sector centerpieces. The cells were scanned at 280 nm or 555 nm every minute and 500 scans were recorded for every sample. Data were analyzed using the program SEDFIT  with the6 equipped JEM-400 transmission electron microscope (JEOL) operated at 120 kV. Images were recorded at a nominal magnification of 60,000x on a 4k X 4k CCD camera F416 (TVIPS), resulting in 0.1890 nm per pixel. Particles were manually selected using EMAN2  and air-sprayed as described  onto freng EMAN2  and furtng EMAN2 .2-373 fusion constructs. Nocodazole (Sigma-Aldrich) was added at 330 nM for 14–16 hr before fixation. Cells were permeabilized with a 0.5% triton-X-100 solution in PHEM  buffer for 2 min and fixed with 4% PFA in PHEM for 15 min. After blocking with 3% BSA in PHEM supplemented with 0.1% triton-X-100 (PHEM-T), cells were incubated at room temperature for 1–2 hr with the following primary antibodies diluted in blocking buffer: anti-Nsl1 ; 1:1000), anti-Hec1 , CREST/anticentromere antibody . After washing with PHEM-T, goat anti-human Alexa Fluor 647 (Invitrogen) and goat anti-mouse Rhodamine Red (Jackson Immuno Research) were used as secondary antibodies. DNA was stained with 0.5 μg/ml DAPI (Serva) and Mowiol  was used as mounting media. Cells were imaged at room temperature using a spinning disk confocal device on a 3i Marianas system equipped with an Axio Observer Z1 microscope (Zeiss), a CSU-X1 confocal scanner unit , 100×/1.4NA Oil Objectives (Zeiss), and an Orca Flash 4.0 sCMOS Camera (Hamamatsu). Images were acquired as z-sections at 0.25 μm, Fluorescence signals were quantified on unmodified 16-bit z-series images using Imaris 7.3.4 32-bit software . After background subtraction, the mean NSL1 or NDC80 intensity was normalized to the respective EGFP intensity mean signal. Converted 8-bit maximal intensity projections of representative cells are shown in U2OS-lacO cells (gift by G. Kops)  were mai In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom, Jon Pines (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor.Thank you for submitting your article "Molecular basis of outer kinetochore assembly on CENP-T" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study the authors have investigated the binding between the CCAN components CENP-T and CENP-C and the outer kinetochore proteins, Ndc80 and Mis12. The authors used recombinant proteins to demonstrate that CENP-T will bind to Ndc80 and to Mis12-Ndc80 after phosphorylation in vitro using Cyclin B-Cdk1. They identify the phosphorylation sites and show which sites are responsible for binding Ndc80 and Mis12. They use a combination of biochemical and electron microscopy assays to estimate the stoichiometry of the complexes generated and present evidence that CENP-T can bind directly to two molecules of Ndc80 via phospho-threonines 11 and 85, and to one molecule of Mis12 via phospho-serine 201. CENP-C can compete for Mis12 binding to this latter site. Many of the main conclusions have been reported recently in other work. For example, two papers from the Westermann lab (PMIDs: 27524485 and 23334295) and a paper from the Fukagawa lab (PMID: 23334297) previously identified the N-terminal Ndc80 binding peptides in CENP-T and its budding yeast equivalent. Thus, the data in The most important new data in this paper relates to the interaction between CENP-T and the Mis12 complex. An interaction with the Mis12 complex had been suggested based on cellular experiments, but a direct interaction has not been shown biochemically. As this interaction appears to be central to building an appropriate outer kinetochore structure on CENP-T, it does have the potential to represent an important new advance.Essential revisions:1) It should be investigated whether S201 is actually phosphorylated in vitro. The only evidence provided for this is a list of peptides identified by mass spectrometry with a designation of which phosphorylated residue was "called" by the search algorithm. For the peptide containing the SVQ site, there are three other residues that are identified as phosphorylated in distinct peptides (suggesting only singly phosphorylated peptide were identified). Based on the way that these searches work, there is potential uncertainty in identifying the specific residue.2) The authors claim that S201 is phosphorylated by Cyclin B-Cdk1 even though the sequence does not conform to the consensus site. In particular S201 is followed by a valine and from the crystal structures of Cdk1 and Cdk2 it is difficult to envisage how this would bind since their active sites have a bulky residue that in almost all substrates requires a proline to for the peptide chain to fit. From the Materials and methods, it appears that the authors used insect cell produced cyclin B-Cdk1 raising the possibility that S201 was phosphorylated by a contaminated kinase. It will be important to eliminate this possibility before publication.3) If this site is phosphorylated, what proportion of the protein is modified at this site in an in vitro reaction? Based on the presented biochemistry and binding assays, it looks like CDK phosphorylation is fully efficient for promoting the interaction with the Mis12 complex. However, as this would be a non-traditional consensus site for CDK, one would expect its phosphorylation to be substantially less efficient than the other sites in CENP-T. A labeling-based strategy to measure the efficiency of phosphorylation at this site would provide allow the authors to assess the potential relevance of this site.4) Is this site phosphorylated in vivo? Multiple high throughput proteomics analyses have identified other phosphorylation sites in CENP-T. However, the serine 201 site that the authors focus on has only been identified in a single study, again suggesting that its phosphorylation is more rare . Ideally, this would include a phospho-specific antibody or additional mass spectrometry analysis.5) If this site is critically relevant, is CENP-T S210A defective in binding to Mis12C and in recruiting Mis12C to kinetochores in human cells? Moreover, what the authors data show is that the serine 201 residue is important for binding to the Mis12 complex. They do not show that the phospho-regulation of this site is key. For example, if they were instead to mutate the neighboring valine or glutamine, this could also block an interaction with the Mis12 complex as they may participate in mediating the interaction. In addition, it would be very useful if the authors could refine the requirements for this binding. All of the binding experiments use an extended portion of CENP-T. If Mis12 complex binding occurs in a limited region (and only requires phosphorylation at the serine 201 residue), they should be able to substantially narrow this binding region.6) The authors show that CENP-C can disrupt the phospho-CENP-T-Mis12C interaction in vitro. Does CENP-T disrupt CENP-C-Mis12C? The authors should determine the affinity between phospho-CENP-T and Mis12C. If they cannot obtain enough phospho-CENP-T for this measurement, they should attempt to narrow down the Mis12C-binding region to a smaller region and perform the measurements with synthetic phospho-peptides as they did with Ndc80C. If CENP-T binds Mis12C with much weaker affinity than CENP-C, Mis12C will prefer to bind to CENP-C at the kinetochore, making it the major receptor for Mis12C.eLife. Your revised article has been favorably evaluated by Anna Akhmanova as the Senior editor and a Reviewing editor.Thank you for resubmitting your work entitled "Molecular basis of outer kinetochore assembly on CENP-T" for further consideration at The manuscript has been substantially improved but there is one remaining issue that needs to be addressed before acceptance:The phosphorylation of S201 by Cyclin B-Cdk1 is a key point of the manuscript. This does not have a proline at position +1 which, according to the crystal structure of the active site, would be incompatible with binding. The evidence that this is phosphorylated by Cyclin B-Cdk1 relies on baculovirus-produced Cyclin B-Cdk1 that could be contaminated by another kinase. Therefore the authors were asked to use bacterial-expressed Cyclin B-Cdk1 to confirm phosphorylation. In the revised manuscript the authors cite the study by Nori Sagata  that showed phosphorylation at a non-SP consensus motif, to which S201 conforms. The problem with this is that the Suzuki et al. study also used baculovirus-expressed Cyclin B-Cdk1, thus the original problem remains unaddressed. As this is a key point of the manuscript the authors do need to show phosphorylation of S201 by bacterially-produced Cyclin B1-Cdk1. Summary:In this study the authors have investigated the binding between the CCAN components CENP-T and CENP-C and the outer kinetochore proteins, Ndc80 and Mis12. The authors used recombinant proteins to demonstrate that CENP-T will bind to Ndc80 and to Mis12-Ndc80 after phosphorylation in vitro using Cyclin B-Cdk1. They identify the phosphorylation sites and show which sites are responsible for binding Ndc80 and Mis12. They use a combination of biochemical and electron microscopy assays to estimate the stoichiometry of the complexes generated and present evidence that CENP-T can bind directly to two molecules of Ndc80 via phospho-threonines 11 and 85, and to one molecule of Mis12 via phospho-serine 201. CENP-C can compete for Mis12 binding to this latter site. Many of the main conclusions have been reported recently in other work. For example, two papers from the Westermann lab (PMIDs: 27524485 and 23334295) and a paper from the Fukagawa lab (PMID: 23334297) previously identified the N-terminal Ndc80 binding peptides in CENP-T and its budding yeast equivalent. Thus, the data in S. cerevisiae, we emphasize that our micrographs of the entire human outer kinetochore capture the CENP-T induced oligomerization of NDC80C in an unequivocal manner  to characterize the role of CENP-T as a MIS12 complex binder in more detail. Major additions include:– A comparison of the sequence surrounding CENP-T S201 with non-canonical CDK1 substrates revealed a conserved (P)xxT/SxxR pattern that was identified as a non- canonical CDK signature in a recent paper , which we now cite. The extended sequence alignment is shown in 184-215, CENP-T192-215 and CENP- T195-215 all bound to MIS12C in a S201 phospho-dependent manner and competed with CENP-C for the binding to MIS12C. This supports the conclusion that the loss of MIS12C binding by the CENPT2-373 S201A mutant represents a direct effect. The binding of the peptides to MIS12C was studied by size-exclusion chromatography , we find that the phosphorylation of CENP-T at position S201 is crucial for the recruitment of MIS12C. These data are shown in – We adopted a – We now show that CENP- phosphorylated at residue S201 not only forms a complex with MIS12C and NDC80C, but also with KNL1C (KNL1 and ZWINT), thus indicating that P-S201 recruits an entire KMN network. These data are shown in – Our recent structure-function analysis of MIS12C revealed a MIS12C mutant that no longer binds to CENP-C . This MIS12C mutant does not bind CENP-T either, supporting the idea that CENP-C and CENP-T bind MIS12C with an at least partly overlapping site. In addition, we provide direct evidence that binding of CENP-T and CENP-C to MIS12 is competitive. These data are included in Below is a point-to-point answer of the referees’ concerns.Essential revisions:1) It should be investigated whether S201 is actually phosphorylated in vitro. The only evidence provided for this is a list of peptides identified by mass spectrometry with a designation of which phosphorylated residue was "called" by the search algorithm. For the peptide containing the SVQ site, there are three other residues that are identified as phosphorylated in distinct peptides (suggesting only singly phosphorylated peptide were identified). Based on the way that these searches work, there is potential uncertainty in identifying the specific residue.We thank the reviewers for raising this point. The other phosphorylation sites within the relevant peptide that the reviewers refer to are T192 and T195. We do indeed observe a mixture of single, double and triply phosphorylated peptides. However, phosphorylation of T195 and S201 are the predominant post-translational modifications of this peptide. A clarification of the identification of single T195, singleS201 and double T195-S201 phosphorylation events is included as 2) The authors claim that S201 is phosphorylated by Cyclin B-Cdk1 even though the sequence does not conform to the consensus site. In particular S201 is followed by a valine and from the crystal structures of Cdk1 and Cdk2 it is difficult to envisage how this would bind since their active sites have a bulky residue that in almost all substrates requires a proline to for the peptide chain to fit. From the Materials and methods, it appears that the authors used insect cell produced cyclin B-Cdk1 raising the possibility that S201 was phosphorylated by a contaminated kinase. It will be important to eliminate this possibility before publication.bona fide Cdk1 substrate. We have also included the phosphorylation control referred to by the reviewer in Figure 2—figure supplement 1.The reviewer raises an important point that had also puzzled us. In the course of the revision, we realized that the CENP-T sequence encompassing CENP-T S201 contains an evolutionary conserved (P)xxT/SxxR pattern that perfectly matches a signature non- canonical CDK site identified in a nice and systematic recent study  . Thus, w3) If this site is phosphorylated, what proportion of the protein is modified at this site in an in vitro reaction? Based on the presented biochemistry and binding assays, it looks like CDK phosphorylation is fully efficient for promoting the interaction with the Mis12 complex. However, as this would be a non-traditional consensus site for CDK, one would expect its phosphorylation to be substantially less efficient than the other sites in CENP-T. A labeling-based strategy to measure the efficiency of phosphorylation at this site would provide allow the authors to assess the potential relevance of this site.As already pointed out in the response to the previous point, the site containing S201 is part of a non-canonical CDK1 site. In this revision, we analysed by mass spectrometry the phosphorylation status of CENP-T when it is part of a CENP-T:MIS12C complex. For this, we separated free CENP-T and CENP-T:MIS12C by size-exclusion chromatography and analysed different fractions by mass spectrometry. This revealed that peptides lacking the phosphorylation at residue S201 are present in the input sample and in free CENP-T, but not in the fraction containing CENP-T:MIS12C complex. . Thus, o4) Is this site phosphorylated in vivo? Multiple high throughput proteomics analyses have identified other phosphorylation sites in CENP-T. However, the serine 201 site that the authors focus on has only been identified in a single study, again suggesting that its phosphorylation is more rare . Ideally, this would include a phospho-specific antibody or additional mass spectrometry analysis.We agree with the reviewer that this is an important point. We pulled-down endogenous CENP-T from HeLa cells and carried out a mass spectrometry analysis. In data summarized and shown in 5) If this site is critically relevant, is CENP-T S210A defective in binding to Mis12C and in recruiting Mis12C to kinetochores in human cells? Moreover, what the authors data show is that the serine 201 residue is important for binding to the Mis12 complex. They do not show that the phospho-regulation of this site is key. For example, if they were instead to mutate the neighboring valine or glutamine, this could also block an interaction with the Mis12 complex as they may participate in mediating the interaction. In addition, it would be very useful if the authors could refine the requirements for this binding. All of the binding experiments use an extended portion of CENP-T. If Mis12 complex binding occurs in a limited region (and only requires phosphorylation at the serine 201 residue), they should be able to substantially narrow this binding region.2-373 is required for the interaction with MIS12C and that a phosphorylated CENP-T2-373 S201A mutant does not bind MIS12C. We now show that synthetic peptides encompassing residues CENP-T184-215, CENP-T192-215 and CENP-T195-215 bind to MIS12C in a manner that depends on S201 phosphorylation. We also show that these peptides compete with CENP-C for the binding to MIS12C. The binding of the peptides to MIS12C was studied by size- exclusion chromatography  showing that the CENP-T T195A mutation, as well as mutations at T11 and T85 that prevent CENP-T:NDC80C interactions, also abolish the recruitment of MIS12C to CENP-T. The basis for this effect of NDC80C-mediated stabilization of MIS12 binding to CENP-T requires further scrutiny.and NDC80C. The results are shown in a separate main figure . Lack of6) The authors show that CENP-C can disrupt the phospho-CENP-T-Mis12C interaction in vitro. Does CENP-T disrupt CENP-C-Mis12C? The authors should determine the affinity between phospho-CENP-T and Mis12C. If they cannot obtain enough phospho-CENP-T for this measurement, they should attempt to narrow down the Mis12C-binding region to a smaller region and perform the measurements with synthetic phospho-peptides as they did with Ndc80C. If CENP-T binds Mis12C with much weaker affinity than CENP-C, Mis12C will prefer to bind to CENP-C at the kinetochore, making it the major receptor for Mis12C.184-215, CENP-T192-215 and CENP-T195-215 peptides and show that the interaction of these peptides with MIS12C depends on the presence of phosphorylated S201. The MIS12C binding affinity of phosphorylated CENP-T192-215 was determined by fluorescence polarization measurements. These data are shown in We thank the reviewers for raising this point. As mentioned above, we now performed a MIS12C binding experiment with CENP-TOur recently described structure-function analysis of MIS12C revealed three aromatic residues in the MIS12 subunit that are important for the orientation of MIS12C’s head1 to the stalk region of MIS12C . Mutation of these residues prevented binding of CENP-C and also prevented a MIS12C-CENP-T interaction. These data further support that CENP-T and CENP-C bind to MIS12C in a similar fashion and are shown in The manuscript has been substantially improved but there is one remaining issue that needs to be addressed before acceptance:The phosphorylation of S201 by Cyclin B-Cdk1 is a key point of the manuscript. This does not have a proline at position +1 which, according to the crystal structure of the active site, would be incompatible with binding. The evidence that this is phosphorylated by Cyclin B-Cdk1 relies on baculovirus-produced Cyclin B-Cdk1 that could be contaminated by another kinase. Therefore the authors were asked to use bacterial-expressed Cyclin B-Cdk1 to confirm phosphorylation. In the revised manuscript the authors cite the study by Nori Sagata  that showed phosphorylation at a non-SP consensus motif, to which S201 conforms. The problem with this is that the Suzuki et al. study also used baculovirus-expressed Cyclin B-Cdk1, thus the original problem remains unaddressed. As this is a key point of the manuscript the authors do need to show phosphorylation of S201 by bacterially-produced Cyclin B1-Cdk1.The revised version of the manuscript was reviewed favourably, but the reviewers raised a residual concern related to the question whether Ser201 is a bona fide CDK1:Cyclin B substrate. The same issue had been raised already during the first round of review:“2) The authors claim that S201 is phosphorylated by Cyclin B-Cdk1 even though the sequence does not conform to the consensus site. In particular S201 is followed by a valine and from the crystal structures of Cdk1 and Cdk2 it is difficult to envisage how this would bind since their active sites have a bulky residue that in almost all substrates requires a proline to for the peptide chain to fit. From the materials and methods it appears that the authors used insect cell produced cyclin B-Cdk1 raising the possibility that S201 was phosphorylated by a contaminating kinase. It will be important to eliminate this possibility before publication.”In response to this concern, we referred to previous work showing that CDK1:Cyclin B can phosphorylate sequences that do not conform to the S/TP consensus provided other features are present, and we noted that these additional features are indeed present at the CENP-T Ser201 site. We also referred to previous work showing that replacement of Pro with Val is the least damaging substitution for the +1 position of the substrate.We note that the reviewers’ argument that the preference for Pro at the +1 position is due to a bulky kinase residue is imprecise. The preference for Pro is determined by a very unusual main chain angle of the activation segment, typical of a left-handed helix. This peculiar conformation forces a main chain carbonyl of the activation segment to point away from the position that would allow it to establish a hydrogen bond with the main chain of the substrate, and specifically between the target S/T and the +1 residue. Pro is insensitive to this penalty, because it does not have a main chain amide, and becomes therefore selected through a “negative mechanism”. The implication is that phosphorylation of other sequences is energetically more costly, and not that it cannot take place due to steric hindrance, as implied by the reviewers. It follows that adequate energetic compensation with surrounding sequences might render other substrates more appealing.eLife web site. The editorial office contacted me to let me know that revisions require that individual text and figure files are uploaded and I inadvertently forgot to include this control. I apologize for that. However, in retrospect, I don’t think that the reviewers would have found this sufficient, because when the manuscript was re-reviewed the issue was identified for being central and the new request above was issued.In addition to discussing this previous evidence, we had also carried out an experiment showing that the migration shift of CENP-T caused by CDK1:Cyclin B phosphorylation is inhibited in presence of RO-3306, a specific CDK1 inhibitor. Regretfully, this experiment, which admittedly answered the reviewers’ concern only indirectly, had been included in a merged manuscript file that I had uploaded on the In response to this, we argue as follows:First, while we also see that the issue of whether CDK1:Cyclin B is the CENP-T Ser201 kinase in vitro is interesting, we don’t see it as a “key point” of our manuscript, which clearly does not aim to address the issue of CDK1 target specificity in any depth. There are several key points in our manuscript, and certainly key in this context is that Ser201 mediates the interaction of CENP-T with the Mis12 complex when phosphorylated, which we prove beyond reasonable doubt. Key would also be to show that this site is a target of CDK1:Cyclin B in cells. We show that the interaction is suppressed before mitosis, arguing that a mitotic kinase does the job. Inhibiting CDK1 in vivo would not take us very far. So this truly important question will remain unanswered until some proper and complex in vivo sensor can be developed. To summarize, we see the question raised by the reviewers as rather marginal in the context of this manuscript.eLife style request, in my opinion, unless the deficiency it addressed was at risk of invalidating the paper’s main conclusions or interpretation, which is clearly not the case here. We also note that we do not have this reagent in our laboratory. We are aware of a previously generated expression system – by Tim Hunt’s laboratory – that made use of the frog kinase (we use human reagents throughout). The paper is already gigantic and it reports a very large body of high quality work, none of which requires, for its interpretation, that the kinase is known with absolute accuracy.Second, the specific request of using bacterially-produced CDK1:Cyclin B1 only appeared after the second review. During the first review, only a generic statement that the issue is given proper attention was raised. As I explained above, we tried to answer this concern by showing that there is substantial prior art. The reviewers did not find our answer sufficient and have now raised a specific suggestion. This may not be an Third, even if we failed to reproduce the phosphorylation with bacterial kinase, this would not prove our results with the insect cell kinase wrong, because there is no proof that the bacterially and insect cell expressed kinases behave in exactly the same way (regardless of possible contaminations). On the contrary, if we reproduced it, we would still be ignorant of the kinase that phosphorylates CENP-T at Ser201 in cells.All this said, we understand that the reviewers put special meaning onto this question. We have therefore added a new figure,


We conclude our experiment by a finding that boldine is cholinesterase inhibitor; however we report significantly weaker inhibition than that suggested in literature. Likewise, we tried to investigate the mechanism of inhibition and completed it with in silico study. Potential toxic effect on cholinesterases in real conditions is also discussed.Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two enzymes sensitive to various chemical compounds having ability to bind to crucial parts of these enzymes. Boldine is a natural alkaloid and it was mentioned in some older works that it can inhibit some kinds of AChE. We reinvestigated this effect on AChE and also on BChE using acetyl (butyryl) thiocholine and Ellman's reagents as standard substances for spectrophotometric assay. We found out IC Cholinesterases are enzymes splitting esters of choline. We distinguish two enzymes belonging to the group of cholinesterases, AChE which is presented in nervous system and terminates neurotransmission and BChE occurring in serum but the particular function of the enzyme remains undiscovered , 2. They Monimiaceae and Magnoliaceae [50 equal to 8.5 μmol/l [50 for both of them. We tried to propose mechanism of interaction of boldine with both cholinesterases as no evidence in literature about this interaction was found. Discussion of potential toxic effect in real conditions is also given. We hypothesize that boldine would serve as a structure for pharmacology research and, furthermore, boldine would act as a multitarget toxin. Unfortunately, data about boldine impact on targeted receptor site are scattered. We decided to investigate interaction between boldine and AChE in higher details than currently available.Boldine, in chemical terminology (S)-2,9-dihydroxy-1,10-dimethoxy-aporphine , is a naoliaceae , 7 and rAcetylcholinesterase human, recombinant , butyrylcholinesterase from human serum , boldine (CAS 476-70-0), acetylthiocholine chloride (ATChCl), butyrylthiocholine iodide (BTChI) 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), and phosphate buffer saline (PBS) pH 7.4 were supplied by Sigma-Aldrich ; ethanol was purchased from Penta .μl DTNB (1 mmol/l), 25 μl AChE, 450 μl PBS, and 25 μl boldine ethanolic solution, and reaction was started by 100 μl of ATChCl (10 mmol/l). Absorbance was measured immediately and then after 2 min of reaction. BChE activity assay was performed as follows: 400 μl DTNB, 25 μl BChE, 450 μl PBS, and 25 μl boldine ethanolic solution, and reaction was initiated by 100 μl of BTChI (10 mmol/l). Difference in absorbance was measured after 2 minutes.AChE activity was assayed, as triplicate, in standard spectrophotometric cuvette in following way: 400 ε = 14,150 l × mol−1  × cm−1 [Ki was then calculated form Michaelis Menten equations.Enzyme activity was calculated from absorbance using extinction coefficient for 5-thio-2-nitrobenzoic acid   × cm−1 . Dixon pUCSF Chimera  was use50 8.5 μmol/l [Ki for boldine and human AChE equal to 372.30 μmol/l and Ki for boldine and human BChE equal to 321.85 μmol/l. Ki obtained from Dixon plot is directly proportional to IC50  and Thr 120 (2.387 Å) were predicted. Stabilization is provided by π-π interaction of Trp 82 and D ring  with Asp 74 and OH group (position 9) was predicted and stabilized via d A ring . Similard D ring . Our resd ring A . Facts a Peumus boldus) [50 values, as 500–1000 mg/kg  is necessary to cause half probability of fatal poisoning [μmol/l of boldine in its peak [μmol/l concentration in the blood or blood plasma. However, higher concentration is needed for significant decay of AChE activity. Thus, even lethal dose is not enough to cause cholinesterase inhibition.Boldine is contained in various plants, widely used in Latin America for decades countries and also exported into Europe   and it ioisoning . Consideits peak , the afo50 equal to 372.30 μmol/l was found out compared to previously published 8.5 μmol/l. Thus, we claim that boldine is a cholinesterase inhibitor but it has very low affinity with the enzyme and its inhibitory effect reaches very low scale. We also investigated the effect on BChE (IC50 321.85 μmol/l) and it was found to be at the same level as that on AChE. We investigated inhibition mechanism of boldine to cholinesterases and proposed inhibition mechanism of boldine to AChE by docking. Results are supported by previously published findings and our experimental data and indicate inhibition via anionic subsite. We also discussed theoretical effect of boldine on AChE in real conditions. Considering the conclusions, boldine appears weak inhibitor of cholinesterases, not well suited for pharmacological research. On the other hand, its ability to inhibit cholinesterases is attributed to its biological effect though the only low manifestation is expected.Effect of boldine on AChE was reinvestigated, and IC


The objective of this study is to investigate the outcomes and safety of using percutaneous anterior C1/2 transarticular screw fixation as a salvage technique for odontoid fracture if percutaneous odontoid screw fixation fails.Fifteen in 108 odontoid fracture patients (planned to be treated by percutaneous anterior odontoid screw fixation) were failed to introduce satisfactory odontoid screw trajectory. To salvage this problem, we chose the percutaneous anterior C1/2 transarticular screw fixation technique in treatment of these patients. The visual analogue score (VAS) of neck pain and Neck Disability Index (NDI) of all patients were scored at pre-operation, 3 months after operation, and final follow-up. Additional, technique-related complications were recorded and collected.P = 0.000) after operation, and no significant differences were found when compared to 93 non-salvage patients who successfully performed the percutaneous anterior odontoid screw fixation. No screw loose or breakage occurred, all of the odontoid fractures achieve radiographic fusion, bony fusion bridge could be observed at the C1/2 lateral articular facet on 9/15 patients.Percutaneous C1/2 transarticular screw fixation was performed successfully in all 15 patients whose odontoid screw fixation failed. No technique-related complications  occurred. The VAS of neck pain and NDI score improved significantly (We suggest that percutaneous anterior C1/2 transarticular screw fixation is a good alternative salvage technique if percutaneous odontoid screw fixation failed, and it is a minimally invasive, feasible, and safe technique. Odontoid fractures are not uncommon injury, accounting for 9–18% of cervical spine fractures , especiaMany posterior surgical techniques were reported in previous literatures –9, incluHowever, anterior odontoid screw fixation is not suitable for all patients. In Grauer type IIC fracture , which iBetween June 2006 and August 2012, 15  in 108 odontoid fracture patients (planned to be treated by percutaneous anterior odontoid screw fixation) were failed to introduce satisfactory odontoid screw trajectory. To salvage this problem, we performed percutaneous anterior C1/2 transarticular screw fixation in treatment of these 15 patients. Eleven cases were type II odontoid fractures and four were shallow type III fractures according to the Anderson and D’Alonzo classification system . The aveThis study was approved by the IRB  of the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, and informed consent was obtained from every participant.We try to perform the percutaneous anterior odontoid screw fixation on all of 108 patients: firstly, the procedure of percutaneous anterior odontoid screw fixation was described by Chi et al. . BrieflyThen, the protective tube  was inserted and the guide tube  was inserted inside of the protective tube, and the guide tube was placed to the anteroinferior of C2 body; a K-wire with a 1.2-mm diameter was drilled into the odontoid process. However, in the 15 patients included in present report, the trajectory of the K-wire was placed unsatisfactorily  of neck pain and Neck Disability Index (NDI) scores were obtained at the time points of pre-operation, 3 months after operation, and final follow-up.CT scans were obtained at the final follow-up to assess the fusion of odontoid fractures and bony fusion bridge at the C1/2 lateral articular facet.t test. The level of significance was set at P < 0.05.The data were collected and analyzed with the software of SPSS . The data of pre-operation, 3 months after operation, and final follow-up were tested by the repeated measures analysis of variance (ANOVA), The comparisons of VAS of neck pain and NDI scores between two groups were tested by independent two-sampled Percutaneous C1/2 transarticular screw fixation was performed successfully in all 15 patients whose odontoid screw fixations failed. The mean operative time was 53 ± 15 min, which was 12 min more than the time cost in the 93 non-salvage patients whom were successfully performed the percutaneous anterior odontoid screw fixation (Table P = 0.000) and maintained at the final follow-up, with 1.29 ± 0.55 (P = 0.420). Similar pain relief was achieved at the 93 non-salvage patients, with 6.29 ± 1.44 at pre-operation to 1.44 ± 1.01 at 3 months after operation (P = 0.000), and maintained at the final follow-up, with 1.36 ± 0.91 (P = 0.451). When compared between the 15 salvage patients and 93 non-salvage patients, no statistically significant difference was found at pre-operation (P = 0.363), 3 months after operation (P = 0.804), and final follow-up (P = 0.738)  and maintained at the final follow-up, with 3.13 ± 2.95 (P = 0.369) in the 15 salvage patients. And similarly, NDI improved from 35.35 ± 5.89 at pre-operation to 4.20 ± 2.90 at 3 months after operation (P = 0.000) and maintained at the final follow-up, with 4.42 ± 2.80 (P = 0.494) in the 93 non-salvage patients. No statistically significant difference was found at pre-operation (P = 0.610), 3 months after operation (P = 0.550), and final follow-up (P = 0.104)  avoid changing patient’s position; (2) avoid other more skin wound; (3) the minimally invasive was similar to the percutaneous anterior odontoid screw fixation only one 10-mm skin wound; (4) good results with a high fusion rate, 15/15 (100%) odontoid fusion, some of the patients with bony fusion bridge observed at the C1/2 lateral articular facet, for some other patients without the obvious bony fusion bridge at the C1/2 lateral articular facet, no screw breakage occurred, and space of the C1/2 lateral articular facet was decreased and tend to grow the bony bridge.Same to some other minimally invasive spinal surgical technique, the percutaneous anterior C1/2 transarticular screw fixation had its limitations. Firstly, the group size is small, because all of the 15 salvage patients are failure of anterior odontoid screw fixation; secondly, the learning curve of this technique was longer than the traditional surgical technique; thirdly, this technique had the potential risk to injure the anterior soft tissue , and our experience was that the tip of the guide tube must be pressed tightly to the front of the C2 body to avoid allowing the soft tissue to enter the space between the tip of guide tube and C2 vertebral body.Percutaneous anterior C1/2 transarticular screw fixation is a good alternative salvage technique if the percutaneous odontoid screw fixation is failure for odontoid fracture. And it is a minimally invasive, feasible, and safe technique.


Thus, phasic inhibition, but not excitation, shapes SWR oscillations in the hippocampal CA1 region in vivo.Sharp wave-ripple (SWR) oscillations play a key role in memory consolidation during non-rapid eye movement sleep, immobility, and consummatory behavior. However, whether temporally modulated synaptic excitation or inhibition underlies the ripples is controversial. To address this question, we performed simultaneous recordings of excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) and local field potentials (LFPs) in the CA1 region of awake mice in vivo. During SWRs, inhibition dominated over excitation, with a peak conductance ratio of 4.1 ± 0.5. Furthermore, the amplitude of SWR-associated IPSCs was positively correlated with SWR magnitude, whereas that of EPSCs was not. Finally, phase analysis indicated that IPSCs were phase-locked to individual ripple cycles, whereas EPSCs were uniformly distributed in phase space. Optogenetic inhibition indicated that PV •High-resolution synaptic current recording during sharp wave-ripples (SWRs) in vivo•Inhibition dominates over excitation during SWRs in the hippocampal CA1 region•Phasic inhibition, but not excitation, is phase-locked to individual ripple cycles•+ interneurons substantially contribute to SWR-associated inhibitory conductancePV + interneurons.Using high-resolution whole-cell recording of postsynaptic current from CA1 pyramidal neurons in awake mice, Gan et al. find that during sharp wave-ripples, inhibition dominates over excitation, is phase-locked to individual ripple cycles, and is primarily mediated by PV Sharp wave-ripple (SWR) oscillations in the hippocampal CA1 region play a key role in memory consolidation during non-rapid eye movement sleep, immobility, and consummatory behavior . DespiteTo probe the synaptic mechanisms of SWR oscillations, we performed simultaneous recordings of excitatory and inhibitory synaptic currents (EPSCs and IPSCs) and LFPs in the CA1 region of awake mice in vivo . Animalss), which on average was 17 ± 1 MΩ in our dataset  and reduced its half-duration  inhibition dominates over excitation; (2) phasic inhibition, but not excitation, is positively correlated with SWR amplitude; (3) phasic inhibition, but not excitation, is phase-locked to individual ripple cycles; and (4) PVrneurons . Thus, t+ interneuron firing in the early ascending phase . PVJ.G. planned experiments, performed experiments, and analyzed data, S.-m.W. performed initial experiments, A.J.P.-A. supervised initial experiments, J.C. and P.J. conceived the project, and P.J. performed simulations and wrote the paper. All authors jointly revised the paper.


Marine mammals are important sources of food for indigenous residents of northern Alaska. Changing sea ice patterns affect the animals themselves as well as access to them by hunters. Documenting the traditional knowledge of Iñupiaq and Yupik hunters concerning marine mammals and sea ice makes accessible a wide range of information relevant to understanding the ecosystem to which humans belong. We interviewed hunters in 11 coastal villages from the northern Bering Sea to the Beaufort Sea. Hunters reported extensive changes in sea ice and weather that have affected the timing of marine mammal migrations, their distribution and behaviour and the efficacy of certain hunting methods. Amidst these changes, however, hunters cited offsetting technological benefits, such as more powerful and fuel-efficient outboard engines. Other concerns included potential impacts to subsistence hunting from industrial activity such as shipping and oil and gas development. While hunters have been able to adjust to some changes, continued environmental changes and increased disturbance from human activity may further challenge their ability to acquire food in the future. There are indications, however, that innovation and flexibility provide sources of resilience. Safe, s2.Interview participants were selected with the assistance of the tribal government in each community as well as community representatives to various co-management organizations responsible for governance of traditional marine mammal hunting . The intTraditional knowledge itself is the product of careful, systematic observation and confirmation by hunters over a lifetime, combined with what has been learned from other hunters and preceding generations . It is sThe research project was approved by the responsible co-management organizations for the different species studied. In recognition of the intellectual property rights of the interview participants, free, prior and informed consent  was obta3.Balaena mysticetus) arriving at Wainwright and Barrow earlier in spring and whaling near Barrow occurring earlier in the spring and later in the autumn. Hunters from Barrow and Kaktovik have noted more bowheads overall during recent decades. Less multiyear sea ice and thinner shorefast ice has made it hard to find ice on which to haul whales out for butchering in spring near Barrow and Wainwright. Bowhead whales no longer appear to feed along the edge of the shorefast ice southwest of Barrow in spring.Notable changes in the behaviour of marine mammals include bowhead whales (Odobenus rosmarus divergens) have hauled out on land near Point Lay in the tens of thousands. Formerly, they were seen on land in summer but not in large numbers. Haulouts on ice, however, are smaller (10–100 s) now than in the past, when a few thousand walruses might be seen together on large ice floes; ice floes in recent years are smaller and thinner and cannot support the weight of so many animals. Walrus body condition is good with no change observed over time.In recent summers, Pacific walruses (Pusa hispida), bearded seals (Erignathus barbatus) and walruses are associated with ice, they also found also in ice-free water. Bearded seals and walruses can sleep on the water; walruses can inflate their necks to float while they sleep. Ringed seals rarely haul out on land, but bearded and spotted seals (Phoca largha) will; sometimes, they are seen together next to passes between barrier islands. Less snow on the ice in Norton Bay has reduced the number of ringed seal dens near Elim. There are now fewer pressure ridges around which snow drifts can form in Kotzebue Sound, though seal holes remain plentiful through the ice in spring both there and in Norton Bay. Young ringed seals were seen on beaches near Wainwright in the summer of 2010 and near Kivalina, Kotzebue and Shishmaref in recent years. These seals appeared to be ill and in poor condition. Barrow hunters reported that some bearded seals had thinner blubber in recent years, which produced different seal oil . Old bearded seals have yellow blubber that makes yellow seal oil; this has not changed with time.Although ringed seals  and species were hunted sequentially. Now, migrations are compressed into a two-week or shorter period in May, so hunters must hunt earlier for multiple species in a shorter period of time. Hunters must also hunt later in the autumn when the sea ice starts to form, but more frequent autumn storms make it harder to do so.Environmental change is not the only factor influencing hunters. There is much concern, for example, about the potential impacts of offshore oil and gas activity as well as increased commercial vessel traffic. Noise disturbs marine mammals, as hunters see from their own activities. For example, increased snowmachine (snowmobile) traffic on sea ice near Barrow is seen as one factor in decreasing numbers of whales near the edge of the shorefast ice.4.Changes in sea ice affect hunters directly by changing access and altering the utility of ice as a substrate for hunting. Changes in sea ice affect hunters indirectly by altering the distribution, timing, behaviour and local abundance of marine mammals. These effects do not occur in isolation, but as a suite of factors that combine to alter hunting behaviour and success.These findings are consistent with other recent studies of traditional knowledge and marine mammals in the region ,13–16 anOur results are also consistent with biological and ecological studies of Arctic marine mammals, many of which describe observed and expected negative impacts to species and those who hunt them, as well as the ways some species appear to be benefiting from less ice ,23. For Information provided by many hunters in many locations observing the environment as it changes and observing marine mammal responses to those changes is well beyond what scientists can do with research projects that are limited to one or two seasons in one location. For example, a review of the scientific literature indicates that bearded seals rarely haul out on land in Alaska ,27. WithThe changes to sea ice that have been observed to date are extensive, and greater decreases are expected in the decades to come . For now


Equus distal forelimb is more complex than traditionally conceived. The current understanding is that the horse distal forelimb consists of one complete digit (III) and two reduced splint metacarpals (II and IV). Metacarpals II and IV each exhibit a ventral ridge, which we suggest represents the undifferentiated digits I and V. These ridges are present in the tridactyl Mesohippus, but are absent in the tetradactyl Hyracotherium. The carpal articulations of the five metacarpals match those of pentadactyl taxa. Distally, the frog, a V-shaped structure on the ventral hoof represents digits II and IV, and the wings and hoof cartilages of the distal phalanx are digits I and V. We relate this revised interpretation of the Equus forelimb to Laetoli footprints, and suggest the Hipparion side impressions are created from the hooves of I and V, rather than from II and IV. We show shades of pentadactyly within the Equus manus.We revisit digit reduction in the horse and propose that all five digits are partially present in the modern adult forelimb. Osteological descriptions of selected tetradactyl, tridactyl and monodactyl equids demonstrate the evolution of the forelimb. Histological, osteological and palaeontological evidence suggest that the  In contrast to the squamates, where entire limbs are lost completely, mammals exhibit a lesser degree of reduction where only individual digits are lost or simplified. In many carnivores, digit I is removed from the locomotory apparatus, but persists on the limb as the dewclaw . Among tEquus. Hyracotherium at the onset of the equid evolution had four metacarpals. The common subsequent main taxon is Mesohippus where the digits are reduced to three. Hyracotherium gave rise to the numerous tridactyl equids that prevailed from the Early Oligocene to the Pleistocene [The horse lineage exhibits the most extreme digit reduction, resulting in the monodactyl forelimb of istocene –7. The aDinohippus, leading to the modern horse [Pliohippus and Astrohippus, single-toed horses that are now extinct [Thoatherium, an extinct ungulate from the Miocene of South America [Monodactyly occurred twice within the equid lineages. The first time it occurred was in the Miocene, starting with rn horse . Monodac extinct . The red America .The reduction of digits in the horse is accompanied by an increase in overall limb length , therefoEquus for the domesticated horse Equus caballus. We suggest changing the identity of certain structures of the manus  of the domesticated horse. Using the osteology, joint articulations, nerve and vessel distribution, and palaeontology of the distal forelimb, we theorize that the Equus manus maintains remnants of the ‘missing’ digits. While it is already known that digits II and IV persist proximally as the splint metacarpals, we propose that digits I and V are also present proximally and that components of all five digits are found distally within the manus.In this study, we use the term Equus. Our study suggests elements of pentadactyly among the most famous single-toed species.We show that the evolutionary change to monodactyly is not as dramatic as previously thought and that the horse forelimb is more similar to that of its pentadactyl, tetradactyl and tridactyl ancestors. Although the modern horse maintains only one complete digit, the identities of all five digits are preserved in both the skeletal and soft anatomy as embedded elements into the dominant digit, and the digit positions are consistent with horses in earlier stages of evolution. We propose an hourglass pattern of reduction where the proximal- and distal-most parts of the digits are retained, the middle regions of the digits are completely reduced. We question the degree of digit reduction in both extinct and extant horses, and rethink the status quo of monodactyly in 2.Equus fetal specimens were obtained from Ward's Natural Science pre-fixed in formalin and frozen. The gestational ages of the fetal specimens were estimated using the femoral length [Two l length  and apprl length . The extEquus specimen was cut in three coronal sections perpendicular to the axis of the distal phalanx, creating three sections. These sections include the proximal end of the distal phalanx including the hoof cartilages, middle end of the distal phalanx including the frog, and distal end of the distal phalanx. An additional adult Equus hoof was sectioned in two coronal cuts, creating two sections. These sections include the proximal end of the distal phalanx and middle end of the distal phalanx near the merging of the frog. These three full-term sections and two adult sections were used to examine the gross morphology of the frog and keratinous hoof.The hoof of a full-term Hyracotherium), tridactyl (Mesohippus) and monodactyl (Dinohippus) equid, as well as the modern horse to delineate the osteology and carpal–metacarpal articulations.We describe the detailed osteology and metacarpal articulations of the horse and several extinct equids using specimens from the American Museum of Natural History, Yale Peabody Museum and Museum of Comparative Zoology at Harvard. From the line of taxa leading to the modern horse, we selected a representative tetradactyl (Hyracotherium as the tetradactyl horse as this represents the onset of the lineage of equids. Mesohippus was chosen as this is an abundant taxon and represents one of the first tridactyl horses. Dinohippus represents the direct ancestor of Equus.We chose Phenacodus). This taxon was chosen as the pentadactyl species as this is a condylarth that is close to the origin of the ungulates. We also describe the proximal carpal–metacarpal morphology of a tridactyl Hypohippus that has a unique specialization among equids. We also describe the Hipparion footprints from Laetoli and the osteology of the distal limb of this taxon.We also describe the articulations of a representative pentadactyl ; crown–rump: 58 cm ;NS 291: no data;NS 292: unossified femoral length: 69 mm ; crown–rump: 58 cm .Using the crown–rump length  and ossi3.2.Hyracotherium and other related archaic perissodactyl taxa from Condylarthra [Hyracotherium during the Eocene there are adaptive radiations . We selected a representative tetradactyl horse (Hyracotherium), tridactyl horse (Mesohippus) and monodactyl horse (Dinohippus) as the primary extinct horses for this study, as well as Hipparion to relate the limb anatomy to the only available fossil footprints from Laetoli.This study includes common taxa that represent the major stages of horse limb evolution. We chose one pentadactyl taxon, 3.3.3.3.1.b and 3a; The distal front limb consists of four complete digits. Metacarpal III is the widest and longest bone . Digit III is dominant; it is has the largest and widest metacarpal. The articular view at the wrist shows three distinct facets corresponding to metacarpals II, III and IV, and large rounded protrusions over digits II and IV. There is a textured region on the proximo-ventral surface of metacarpal II that splays outward and tapers distally into a thin ridge that extends down the shaft of the metacarpal. The ridge is distinct from the shaft of metacarpal II. Metacarpal IV is similar to metacarpal II in length and width. In some specimens, metacarpal V presents as a short, proximal, tubular protrusion on the ventral surface of metacarpal IV. In specimens where this rudiment is absent, there is a distinct ridge with a fusion line on the ventral surface of metacarpal IV, which extends almost completely down the shaft. The ridges on the surfaces of metacarpals II and occasionally of IV of Mesohippus resemble those of the modern horse.The forelimb consists of three digits , each with a complete metacarpal, proximal, middle and distal phalanx figures c and 3b.The phalanges of the dominant digit are larger, fuller and wider than those of the side digits. The dominant distal phalanx is a single, elongated, tubular-shaped bone. In dorsal view, the bone is somewhat narrow and spade-shaped, with a deep median split on the terminal edge. There is a constriction in solar view separating the distal phalanx proper from the angles. In lateral view, the distal phalanx is shallow, and there are no medial or lateral wings above the angles. The side proximal and middle phalanges of digits II and IV are thin and complete. Laterally, each distal phalanx has a short blunted angle with no wing above it.3.3.3.Equus (figures f and 3c). There is one dominant central digit III and two lateral splint bones . Metacarpals II and IV are full proximally and taper distally. On both metacarpals II and IV, there is a slightly expanded, textured area at the proximo-ventral area. This textured surface extends distally into a ridge at the ventral surface that gradually narrows. There is a faint depression on the outer surface of the ridge that separates the ridge from the remainder of the shaft. The distal aspect of metacarpals II and IV tapers into a thin rounded point.The metacarpals appear to be isometrically smaller than those of  figures f and 3c.There is one complete digit consisting of a proximal, middle and distal phalanx. The distal phalanx is a wedge-shaped bone that appears crescentic in dorsal and solar views. There is a small midline split on the terminal edge of the distal phalanx, which is most visible in dorsal view. The angles are present, but blunted. The wings are large, boxy and separated from the angles by a wide groove. The wings in dorsal view flare outward. The wings are separated into parietal and inner surfaces by a shallow groove.3.3.4.The proximal row of carpals consists of four bones, and the distal row contains three to four bones, with the trapezium being variably present figures  and 5. TThere are three phalanges on the functional manus: proximal (long pastern), middle (short pastern) and distal (coffin). The distal phalanx is single and wedge-shaped with a crescentic terminal edge that forms a broad symmetrical ‘U’ without a median split. In solar view, the crescentic surface terminates in two symmetrical pointed knobs, termed the angles, with one medial and the other lateral.Above the angles there are two symmetrical dorsal protrusions named the wings, which are positioned medially and laterally. The wings are triangular/wedge-shaped and are pointed dorsally, paralleling the shape and orientation of the extensor tubercle. The size of the wings varies between individuals. The wings are separated into a parietal and inner surface by a deep groove, which houses a portion of the medial and lateral hoof cartilages. The hoof cartilages protrude behind the distal phalanx, and on the internal aspect, there are numerous ligaments.3.4.3.4.1.a and Metacarpal–carpal: Metacarpal I articulates laterally with metacarpal II  and proximally with the trapezium (one large facet) figures a and 6. Carpal–carpal: The trapezium articulates dorsally with the trapezoid  and proximally with the scaphoid . The trapezoid articulates laterally with the magnum  and proximally with the scaphoid (one large facet). The magnum articulates laterally with the unciform (one large facet) and proximally with the lunate (one large facet). The unciform articulates proximally with the lunate  and triquetrum (one large facet). The scaphoid articulates laterally with the lunate . The lunate articulates laterally with the triquetrum . The triquetrum articulates ventrally with the pisiform (one large facet).3.4.2.Phenacodus are present, aside from the articulations of metacarpal I, as this digit is absent in Hyracotherium , and proximally with trapezoid (one large facet) and magnum  figures f and 6. Hyracotherium and Mesohippus are noted, aside from the apparent lack of trapezium in the examined Dinohippus specimens.Carpal–carpal: The same articulations as in 3.4.5.Dinohippus are present, with the addition of an articulation between metacarpal II and the trapezium, when present . Metacarpal III is wider and slightly longer than the side metacarpals, which are thin, elongated and symmetrical. Rudiments of both metacarpals I and V are seen at the proximal shafts. These rudiments are approximately 1 cm long and are positioned on the ventral surfaces of metacarpals II and IV, respectively.There is one dominant metacarpal (III) and two side digits (II and IV), which are complete with a metacarpal, as well as with proximal, middle and distal phalanges d. Metaca3.6.3.6.1.a,b and The frog is a dense fibrous structure that contacts the ground figures a,b and 83.6.2.a–c). The dorsal surface of the hoof has a keratin-generating region proximally. The numerous layers of keratinous lamellae grow distally over the dorsal surface of the distal phalanx. Between the dorsal surface of the distal phalanx and the hoof keratin is the nail bed , which also has germinating properties. This structure adheres to the dorsal periosteum of the distal phalanx. There are additional generating regions on the ventral surface of the hoof, forming ventral lamellae. The white line (a tubular horn between the lamellae) is formed by the junction of the dorsal lamellae and the ventral lamellae. In the human nail, the nail bed terminates at the apex [Equus, there is additional hoof (nail) material extending ventral to the apex, growing from a nail bed on the ventral distal phalanx as well as on the frog. There are under-folds of keratin at the lateral and medial aspects of the proximal hoof, which are distinct from the frog and the sole of the hoof proper. These under-folds are positioned in the region ventral to the medial and lateral hoof cartilages. On the ventral surface of the hoof, there are five regions, each with their own orientation of lamellar fibres; two sections are under the frog, two are at the medial and lateral under-folds, and the fifth and largest region comprises the remaining hoof surface towards the crescentic edge.There are several parts to the keratinous horse hoof a–c. The the apex , whereasd–f).The hooves of fetal specimens are softer than those of adult individuals. Externally, our horse fetus exhibits four distinct infoldings on the ventral region hoof, whereas the dorsal region of the hoof is smooth without subdivisions. These folds are distinct distally and blend together proximally towards the middle phalanx d–f.3.7.Hipparion [Equus, there is a single impression that is rounded anteriorly, with a V-shaped indentation at the posterior aspect. In the tracks of Hipparion, the anterior-most aspect of the print terminates in a sharp median point, there is no V-shaped impression posteriorly, and there are two additional side impressions slightly posterior to the dominant impression. In addition, they also show a large round impression behind the hoof , including the tracks of ipparion . These tthe hoof .Figure Hipparion forefoot was tridactyl, with digit III being dominant, and digits II and IV as the fully developed side limbs (e). The forelimb anatomy closely parallels that of the other tridactyl equids, including our previously described Mesohippus limbs. The Hipparion distal phalanx exhibits well-developed wings above the angle. These wings are oriented horizontally and are positioned close to the solar surface of the distal phalanx, while the angles are blunted. There is a deep cleft at the median plane of the terminal edge of the distal phalanx. The side digits are thinner and shorter than the dominant digit. The side distal phalanges lack the wings seen in the dominant distal phalanx, but possess a pronounced single angle on the outer edge of the hoof.The de limbs e. The fo3.8.a)Fourteen distinct nerve bundles characterize the proximal end of the horse manus. We find two to four nerve bundles positioned dorsal to metacarpal III, one nerve bundle adjacent to the inner surface of each of metacarpals II and IV, and one to two nerves adjacent to the outer surface of metacarpals II and IV. In addition, there are three to four large nerve bundles located ventrally in the proximity of the muscle groups, and an additional small nerve bundle is situated in the region ventral metacarpal III. In this area, there are two to three distinct bundles of associated vein, artery and nerve (VAN) around metacarpal II and two VANs around metacarpal IV aFigure b).Approximately three-quarters of the way down the metacarpal, 8 to 10 nerves are found. Two to three nerves are positioned dorsal to metacarpal III, and the remaining nerves are positioned ventrally. There are two large nerve bundles ventrally, one lateral and one medial. Three smaller nerve bundles are seen adjacent to the distal end metacarpal II and two bundles are found adjacent to the distal end of metacarpal IV. We find five distinct VANs; one to two large VANs ventrally , three smaller VANs in the ventral region and zero to one VANs dorsally b.c).At the cross section through the proximal phalanx, 9 to 10 nerves are seen, with one to two nerves positioned dorsally to the proximal phalanx. There are two large nerve bundles on the ventral region, one lateral and one medial. There are four intermediate-sized nerve bundles, with two positioned laterally and two medially, which are relatively symmetrical in position. We find two additional small nerve bundles ventrally, one lateral and one medial. We find five to six VANs total in this region. We observe two large VANs . There are one to two additional smaller VANs laterally, one small VAN medially and one VAN dorsally c.d).A transverse section through the middle bony phalanx reveals 9 to 12 nerves, which are overall smaller than the proximal counterparts. We find one to three small nerve bundles positioned dorsally to the middle phalanx, and the remaining nerve bundles are positioned ventrally. In the ventral region, there are two, symmetrical, intermediate-sized nerve bundles, one lateral and one medial. There are several nerves surrounding the lateral hoof cartilage. We find two large VANs ventrally , and four to five additional smaller VANs surrounding the middle phalanx d.e).At the most distal section, five to six nerves are identified, with two to four nerves surrounding the dorsal aspect of the distal phalanx. We find two to three additional nerve bundles are positioned just ventral to the distal phalanx, one to two laterally and one medially. We find a single VAN triad ventral to the distal phalanx, and numerous minute arteries throughout the section. There is a reduction in large nerve number from the proximal counterparts. In this region, the nerves are numerous, but the size is notably reduced, possibly reflecting significant branching of the more proximal nerve segments e.4.Limb reduction has occurred several times during tetrapod evolution . This caTetrapod forelimbs are organized into the stylopod (humerus), zeugopod (radius and ulna) and autopod segments  . During Equus retains the identities of digits I–V, most notably at the wrist and distal hoof , and in Dinohippus, a faint suture line is visible at the outer surface. We believe these fusion lines indicate that the ridges are separate structures from the splint metacarpals, digits I and V, that throughout time have embedded onto the surfaces of digits II and V. In Hyracotherium, where digits II–V are fully present, only metacarpal II possesses a clear ventral ridge. This resembles the elevation seen on metacarpals II and IV in Equus, where it is expanded and triangular proximally and tapers distally into a thin ridge. The lack of ridges on the other metacarpals of Hyracotherium reinforces this hypothesis, as all other digits are already present. Metacarpals III–V of Hyracotherium are, therefore, singular and do not contain remnants of other digits, as is seen in later equids.Side metacarpals II and IV have a ridge on the ventral surface, which is a shared feature of the extinct monodactyl ese taxa . In Mesohe ridge b, and inHypohippus, where all five digits are present proximally towards the wrist. In Hypohippus, metacarpals I and V are present at the proximal shaft, as ovoid bony elements positioned on the ventral surface of metacarpals II and IV. These rudiments of the incomplete metacarpals I and V are in the same position as the ventral surface irregularities in Equus, Dinohippus and Mesohippus. This further supports the identities of the ventral ridges and textured regions in the monodactyl and tridactyl horses as lost digits I and V. The digit identities of the fragments as I and V in Hypohippus are confirmed by carpal articulations; the rudiment of I articulates with the trapezium, and the rudiment of V articulates with the unciform.This proposed location of metacarpals I and V is consistent with the extinct tridactyl Equus hand reflects the evolutionary history. In the tetradactyl Hyracotherium, the four complete digits are not aligned horizontally, but instead curve inward, creating a tubular structure. This orientation remains consistent in the tridactyl Mesohippus manus, where side digits II and IV arch inward in relation to dominant digit III, and the remnants of digits I and V are positioned ventral to the side digits. In the modern horse, the manus maintains this overall curved orientation created by splint digits II and IV and their ventral ridges representing reduced digits I and V. Therefore, the transition from the tetradactyl equids to the modern horse was not as dramatic as previously thought.Interestingly, despite a reduction in the number of complete digits and an increase in their size, the overall positions of individual elements and their borders in the manus has remained consistent throughout equid evolution. We see evidence of this evolutionary history in the anatomy of the modern horse forelimb. The horse carpus and manus retains the same borders of individual bones that were seen throughout equid lineages. Moreover, the orientation and general structure of the Equus is small and variably present [The trapezium of  present ,31, wher present –34. We bEquus manus, the carpal–metacarpal articulations are consistent with those in Phenacodus, a primitive pentadactyl taxon  articulates with the trapezoid and occasionally ventrally with the trapezium. In Phenacodus, metacarpals IV and V articulate with the unciform, which is consistent with our proposal that the lateral splint bone, which articulates with the unciform, represents metacarpals IV and V. The metacarpal–carpal relations have remained the same regardless of apparent digit reduction in the horse, supported by the similarities in articulations between the ‘monodactyl’ Equus and the pentadactyl Phenacodus distal forelimbs.Using our proposed identities of the five digits in the  figures a and 4. Hyracotherium has a difference in the number of digits in the forelimb versus the hindlimb. In Dinohippus and Equus, both the forelimb and hindlimb are monodactyl, and in Mesohippus, the front and back limbs both consist of three complete digits. In Hyracotherium, the forelimb is tetradactyl and the hindlimb is tridactyl. Interestingly, the Hyracotherium foot possesses small splint digits on the ventral surfaces of metatarsals II and IV, which represent ‘missing’ metatarsals I and V [Hyracotherium hindlimb foreshadows the rudiments in the hands of the later equids; Mesohippus and Hypohippus possess elements of metacarpals I and V on the ventral surfaces of complete digits II and IV, respectively. Unlike the limbs of Hyracotherium, the forelimbs and hindlimbs of the other described equids are notably similar, both in number of digits and overall morphology.Among the extinct equids described, only  I and V ,36. The Equus. The distal bony phalanx of the hoof is digit III. On the medial and lateral sides of the distal phalanx, there are firmly attached small bony thickenings termed the wings, which resemble small distal phalanges but appear to be fused with the bony distal phalanx proper. We propose that the wings and hoof cartilages represent the distal aspects of digits I medially and V laterally. These wings are present in the tridactyl taxon Hipparion, where digits II–IV are fully formed, and are absent in the tetradactyl Hyracotherium, which only lacks digit I. We expected to find a medial wing on the distal phalanx of Hyracotherium, representing the single missing digit, but this taxon shows no apparent evidence of the pollex distally. We suggest that the presence of these ‘miniature hooves’ in Equus and Hipparion indicates that digits I and V are partially expressed distally, and are not absent as previously suggested. Correspondingly, on the hoof region directly ventral to the cartilages, there are distinct areas each with their own corium and keratinous lamellar fibres, which we propose represent the hooves of digits I  and V .We also find morphological evidence for partially developed digits I, II, IV and V distally on the phalanx and hoof of Mesohippus and the other tridactyl horses. The frog is absent from other living perissodactyls, including the tapir and rhino, as these taxa both have digits II and IV fully expressed [Equus contains fibrous material (cushion) surrounded by keratinous lamellae that are distinct from the fibres of the hoof proper. The frog has its own germinal matrix, corium and papillae [The metacarpals of digits II and IV are located ventral to metacarpal III. If these digits were to continue distally, their projected position would be ventral to the dominant distal phalanx, consistent with the location of the double-sided frog. Embryonic studies of the distal horse hoof demonstrated regions of apoptosis surrounding the precursors for digits II and IV . We findxpressed . Internapapillae . We propOn the ventral surface of the hoof, there are five distinct regions, each with their own orientation of lamellar fibres. The five regions on the ventral surface of the hoof represent the partially differentiated hooves of all five digits. The medial and lateral under-folds are positioned directly ventral to the medial and lateral hoof cartilages (proposed digits I and V), respectively. These under-folds represent the hooves of these digits. Centrally, there are two additional keratin regions with distinct fibre orientation positioned directly under the frog; these represent the hooves of digits II and IV. The remainder of the ventral hoof is singular and without subdivisions, and is fused to the solar surface of distal phalanx III. This represents the hoof of the main digit III. These five regions each have a distinct orientation of lamellar fibres from each other and separate areas of corium and papillae. The positions of these hoof subdivisions are consistent with our proposed position of digits I, II, IV and V both proximally and distally, where these digits are compressed onto the ventral aspect of the dominant digit.d–f). The common number of ‘petals’ is four on the ventral side and one on the dorsal. The external appearance of the hoof parallels our observation of the adult hoof subdivisions. Ventrally, the four folds of hoof material represent the compressed digits I, II, IV and V, and the single dorsal region represents the hoof of digit III. The ‘lost’ digits are visible in the horse embryo as they are in the adult individuals, and again these digits are positioned ventral to dominant digit III. Most distally these five petals, although they externally display distal borders, merge into a single undifferentiated cone, which extends further and terminates into a notably long and curved point like a median claw. In the literature, these have been termed ‘soft cellular mass’ [In late-term fetuses, the keratinous hooves resemble an unopened tulip flower d–f. The ar mass’ . As the We are proposing that all five digits are present proximally and distally on the horse and ancestors, and that the middle portion of the shaft is absent. This hourglass pattern of reduction where the mid-portion is lost also occurs in fibular reduction in certain artiodactyls. In Pecora, the proximal fibula is present and fused to the lateral tibial condyle, and the distal fibula is represented in the malleolus. In these taxa, the middle portion of the fibula is completely reduced . The batHipparion sp. indicate a large difference in the distal limb anatomy from Equus. The footprints of this taxon were interpreted to indicate a ‘running walk’ gait, different from the modern horse [Hipparion impressions, the anterior aspect is pointed at the midline and there are two smaller side impressions medial and lateral to the main print. Studies have suggested that the side impressions represent the complete splint digits (II and IV) [Hipparion are shorter than digit III [Hipparion skeletal specimens are positioned just above the solar surface of the dominant distal phalanx, and could plausibly leave impressions in the mud if enveloped in their own keratinous hoof material.The Laetoli footprints of rn horse . A typic and IV) ,41,42. Higit III , and phyigit III . We propHipparion footprints lack the V-shaped posterior impression that characterizes the footprint of the modern horse. This posterior impression in the horse is created by the frog, which we propose represents distal digits II and IV. The Hipparion print lacks this V-shaped posterior impression, indicating the absence of a frog in this taxon. In Hipparion, digits II and IV are fully formed, and therefore, it should not have a frog. In place of the position of the frog, the Hipparion footprints exhibit a large round impression, which was hypothesized as mud stuck behind the hooves [The Laetoli e hooves . We prope hooves .Hipparion. The distal phalanges in hipparions and most other extinct equids exhibit a distinct median cleft on the terminal edge, creating an interruption in the U-shaped arc. A keratinous claw fused to the main hoof would explain the presence of this cleft. Such a cleft is present throughout most Equinae and Anchitheriinae. The midline cleft on the distal hoof is absent in adult horse specimens. We observe, however, the presence of the claw in fetal specimens of Equus, which is worn off before horses reach adulthood.We interpret the anterior point in the Laetoli footprints as the presence of a midline claw in Just as the skeletal and hoof anatomy shows hints of the ‘missing digits’, the nerves of the horse show a pattern more similar to the pentadactyl forefoot anatomy, and are not reduced enough to reflect a single digit. At cross-sectional view through the five metacarpals, humans possess approximately 9 to 15 nerves ,45; the The human finger exhibits two large triads of vein, artery and nerve (VAN) per finger, each consisting of a proper palmar digital artery, proper palmar digital nerve and associated vein . These tHorses exhibit congruency in the metacarpal muscle attachments when compared to pentadactyl taxa ,47, and Limb reduction has been extensively studied in squamates –51. The d–f and 11f–h). In the adult, our proposed position of these digits remains ventral to digit III both proximally at the wrist and distally towards the distal phalanx, retaining their embryonic relations.We find the horse distal forelimb shares features with that of an early horse embryo. Horse embryos at several stages  show one, long, cylindrical dominant digit and two splint metacarpal digits, which commence as cartilaginous precursors that ultimately ossify . Skeleta75.Phenacodus. The tridactyl equid Mesohippus shares the ventral ridges on the side digit bones, many of which appear to have a distinct fusion line, and exhibits a lesser degree of digit reduction than previously thought. The frog is a double structure unique to the modern horse, which is newly theorized to represent partially differentiated distal digits II and IV. The wings and hoof cartilages of the distal phalanx represent distal digits I and V. The frog and hoof wings each exhibit regions on the hoof with distinct keratinous lamellar fibres and germinating properties, further supporting our proposal that they represent additional digits. We reinterpret the famous Hipparion Laetoli footprints as exhibiting side expansions of the hoof wings (proposed digits I and V), rather than impressions from the complete side digits II and IV, and suggest the presence of a median claw in extinct equids, explaining the midline cleft on their distal phalanges. The nerves and arteries are more numerous than expected for a true mondactyl taxon, and better reflect the pentadactyl forefoot anatomy. Our study shows anatomical evidence for the presence of digits I and V proximally, in addition to the previously interpreted digits II, III and IV in the horse manus. In addition, we find remnants of digits I, II, IV and V distally surrounding dominant digit III. Our study suggests evidence of pentadactyly hidden within the single complete digit of Equus.The morphology of the distal forelimb in the horse suggests an altered paradigm of monodactyly, in which remnants of the additional four digits are present both proximally and distally. The dominant digit remains as III. The splint metacarpals, previously interpreted as reduced digits II and IV, also contain remnants of digits I and V compressed as ridges onto the ventral surface. Our proposed locations of the proximal metacarpals are confirmed by their carpal articulations, which are consistent with those of the pentadactyl


Tenebrio molitor antifreeze protein (TmAFP) was simulated with growing ice-water interfaces at a realistic melting temperature using TIP4P/Ice water model. To test compatibility of protein force fields (FFs) with TIP4P/Ice water, CHARMM, AMBER, and OPLS FFs were applied. CHARMM and AMBER FFs predict more β-sheet structure and lower diffusivity of TmAFP at the ice-water interface than does OPLS FF, indicating that β-sheet structure is important for the TmAFP-interface binding and antifreeze activity. In particular, CHARMM FF more clearly distinguishes the strengths of hydrogen bonds in the ice-binding and non-ice-binding sites of TmAFP than do other FFs, in agreement with experiments, implying that CHARMM FF can be a reasonable choice to simulate proteins with TIP4P/Ice water. Simulations of mutated TmAFPs show that for the same density of Thr residues, continuous arrangement of Thr with the distance of 0.4~0.6 nm induces the higher extent of antifreeze activity than does intermittent arrangement of Thr with larger distances. These findings suggest the choice of CHARMM FF for AFP-TIP4P/Ice simulations and help explain the relationship between Thr-residue arrangement and antifreeze activity. Antifreeze proteins (AFPs), which consist of polypeptides with various sizes and structures, are found in arctic or antarctic organisms such as bacteria , fungi , plants The DeVries group pioneered experimental and theoretical studies on the interactions between AFPs and ice-water interfaces. They found that AFPs adsorb to ice surfaces and inhibit ice growth through a noncolligative process, which was explained by a mechanism of the melting-point depression on the curved ice-water interface, called the “Gibbs-Thomson” (or Kelvin) effect , 14, 15.To support and complement experimental observations, molecular dynamics (MD) simulations have been performed. McDonald et al. simulated a winter flounder AFP (wfAFP) with pyramidal planes, showing the adsorption of wfAFP to ice surface because of the interactions between Threonine (Thr) residues and oxygen atoms in the ice lattice . Cheng aAs a further step toward understanding the effect of Thr arrangement on antifreeze activity, we here report all-atom MD simulations of Tenebrio molitor AFP (TmAFP) with ice-growing surfaces at a realistic melting temperature using TIP4P/Ice water model . We firsAll simulations and analyses were performed using the GROMACS5.1.4 simulation package –42. The +) were added to establish electro-neutrality in a periodic box of size 5.1 × 12.6 × 8.6 nm3  of TmAFP and its initial COM as a function of time. In To confirm these different extents of the TmAFP-ice binding, diffusion coefficients of TmAFPs were calculated by obtaining the slopes of the mean-square displacements (MSD) of the COM of TmAFP for the initial 0~30 ns) and final 90~120 ns) simulation times. Note that the MSD usually needs to be corrected for finite size effects using Yeh and Hummer’s analytic formula for cubic boxes , but for0 ns simu0 ns and To understand this dependence of the antifreeze effect on FFs, secondary structures of TmAFP were calculated using the DSSP program . Table 2Experimental results have shown that the ice-binding site of AFP consists of many Thr residues and thus forms more hydrogen bonds with the surrounding water than does the non-ice-binding site of AFP , implyinExperimentally, Graether et al. showed that the mutation (Thr to Leu) of the ice-binding site of AFP significantly reduces antifreeze activity , which wAs discussed above, antifreeze activity of TmAFP is influenced by the arrangement of Thr residues. Since Thr residues of the ice-binding site form strong hydrogen bonds with the ice-water interface and induce antifreeze activity, the strength of the AFP-ice binding may depend on the arrangement of Thr residues, although their relationship has not been systematically studied. To resolve this, we calculated minimum distances between Thr residues. In These simulations show the effect of different FFs on the secondary structure and antifreeze activity of TmAFP, and the strength of the TmAFP-ice surface binding, suggesting a choice of CHARMM FF especially for simulations of AFP and TIP4P/Ice water at a realistic melting temperature. In particular, simulations show the higher extent of antifreeze activity when Thr residues are more densely distributed with the distance of 0.4~0.6 nm, indicating the effect of Thr-residue arrangement on hydrogen-bond interactions and antifreeze activity. Note that different types of AFPs have specific binding affinities with various ice-plane types, so our results should be considered for specific AFP and ice plane with caution. To more precisely determine the antifreeze activity of different TmAFPs, it would obviously be interesting to calculate the free energy of binding at the AFP-ice interface, which we hope to report on elsewhere.We performed MD simulations of TmAFP with growing ice-water interfaces using TIP4P/Ice water that can reproduce a realistic melting temperature while still being computationally less expensive than TIP5P or six-site water. Since the interactions between proteins and TIP4P/Ice water have not been verified, we first tested compatibility of TIP4P/Ice water with different FFs such as CHARMM, AMBER, and OPLS. TmAFP binds to the ice-water interface, induces curvature of ice surface, and suppresses ice growth, although their extents differ. CHARMM and AMBER FFs predict more β-sheet structure and lower diffusivity of TmAFP at the ice-water interface than does OPLS FF, indicating the stronger TmAFP-ice surface binding and antifreeze activity. In particular, analyses of hydrogen-bond lifetime show that CHARMM FF more clearly distinguishes the strengths of hydrogen bonds in the ice-binding and non-ice-binding sites of TmAFP than do other FFs, which favorably compares with experiments, implying that CHARMM FF can be reasonably chosen to reproduce experimental observations on the secondary structure of AFP and its interaction with TIP4P/Ice water.To understand the relationship between Thr-residue arrangement and antifreeze activity of AFP, mutated TmAFPs were generated by replacing some of Thr residues with Leu and simulated with TIP4P/Ice water using CHARMM FF. Simulations of mutated TmAFP show that when densities of Thr are same, densely distributed arrangement of the distance of 0.4~0.6 nm more effectively induces antifreeze activity than does intermittent arrangement of Thr with larger distances. This indicates that distances between Thr residues must be small enough to maintain ordered structure of ice-surface water that can form persistent hydrogen bonds with TmAFP. These findings suggest that CHARMM FF can be reasonably chosen for simulations of AFP with TIP4P/Ice water at a realistic melting temperature, as well as help explain experimental observations regarding the difference of the binding strength in the ice-binding and non-ice-binding sites of TmAFP, and the dependence of antifreeze activity on the distance between Thr residues and hydrogen-bond interactions with ice surfaces.