Studies of VASP phosphorylation revealed that this was because of an increase in PKA activity following treatment with PPARγ receptor agonists.Treatment of platelets with PPARγ agonists, identifies a negative regulatory role for PPARγ agonists in the control of integrin αIIbβ3 outside‐in signaling, and provides a molecular basis by which the PPARγ agonists negatively regulate platelet activation and thrombus formation.This study provides further evidence for antiplatelet actions of  Platelets play a vital role in the prevention of blood loss following vascular injury. Through a balance of inhibitory and activating signals, circulating platelets are maintained in a quiescent state in the undamaged circulation and are only activated when signals such as exposed subendothelial matrix proteins at sites of vascular injury outweigh endogenous inhibitory signaling. Platelets adhere rapidly to the site of injury and become activated, characterized by granule secretion, formation of thromboxane A2 and activation of integrin αIIbβ3, which is converted from a low to a high‐affinity conformation, enabling it to bind to fibrinogen and von Willebrand factor (VWF). This initial activation is followed by platelet shape change and aggregation that is supported by fibrinogen binding to the high‐affinity integrin αIIbβ3. Formation of a fibrin coat and clot retraction, which is driven through integrin αIIbβ3‐stimulated outside‐in signaling, then stabilizes the growing thrombus Inhibitory mechanisms are essential for preventing the formation of unwanted thrombi and for limiting thrombus growth and size. Platelets are inhibited through a number of endogenous mechanisms, including by nitric oxide and prostacyclin released from the healthy endothelium We have previously observed the ability of PPARγ agonists to interfere with GPVI‐proximal signaling, but this cannot explain the PPARγ agonist‐dependent inhibition observed following stimulation of platelets with thrombin and other GPCR agonists. Because the integrin αIIbβ3 outside‐in signaling pathway shares several components with the GPVI signaling pathway The results described here support a role for PPARγ in the negative regulation of integrin αIIbβ3 outside‐in signaling through the up‐regulation of PKA activity and subsequent inhibition of phosphorylation of β3 and downstream components of the integrin αIIbβ3 signaling pathway.Fibrinogen, bovine thrombin, H89, GW9662 and IMBX were purchased from Sigma Aldrich . 15dPGJ2 was purchased from Enzo Life Sciences and Ciglitazone and SQ22536 from Tocris Bioscience . The cAMP ELISA kit was from Enzo Life Sciences . Primary anti‐ FAK  (C20), Syk (N‐19), PLCγ2 (Q20), β3 (C20) and actin (C11) antibodies were purchased from Santa Cruz Biotechnology . Phospho‐specific primary antibodies for β3 Y779 and Akt S473 were from Abcam . Anti‐phospho–PKC (protein kinase C) substrate, phospho‐S157 and S239 VASP and phospho‐Ser 19 myosin light chain antibodies were purchased from New England BioLabs , and anti‐phospho‐Tyr 4G10 antibody was purchased from Millipore . Fluorophore conjugated secondary antibodies, Fluo‐4 calcium indicator dye and Alexa‐488 and Alexa‐647 conjugated phalloidin were purchased from Life Technologies . All other reagents were from previously described sources m Na3C6H5O7, 113.8 mm glucose, 72.6 mm NaCl and 2.9 mm citric acid [pH 6.4]). Washed platelets were prepared by centrifugation at 100 × g for 20 min, followed by centrifugation twice at 1000 × g for 10 min in the presence of 1.25 μg mL−1 prostacyclin (PGI2) as described previously m NaCl, 0.34 mm Na2HPO4, 2.9 mm KCl, 12 mm NaHCO3, 20 mm N‐2‐hydroxyethylpiperazine‐N‐2‐ethanesulfonic acid, 5 mm glucose and 1 mm MgCl2, pH 7.3) and rested for 30 min at 30 °C before use.Human blood was obtained from consenting aspirin‐free healthy volunteers, in accordance with the procedures approved by the University of Reading Research Ethics Committee. Blood was collected into 4% (w/v) sodium citrate before mixing with acid citrate dextrose (29.9 m7 mL−1), pretreated with PPARγ agonists or vehicle control (0.1% v/v dimethylsulfoxide [DMSO]), were exposed to fibrinogen (100 μg mL−1) coated coverslips  and incubated for 45 min at 37 °C. Non‐adherent platelets were removed and coverslips washed with phosphate buffered saline (PBS) before fixing using 0.2% (v/v) paraformaldehyde solution. Platelets were permeabilised in 0.1% (v/v) Triton‐X100 prior to staining with Alexa 488 conjugated‐phalloidin for 1 h. Adherent platelets were then imaged with a 100x magnification oil immersion lens on a Nikon A1‐R confocal microscope. Adhesion and spreading data in each experiment were measured by counting the number of platelets and the extent of spreading in five fields of view chosen randomly from each sample.Washed platelets (2 × 108 mL−1 were added to aggregometer tubes in the presence of 2 mg mL−1 fibrinogen and 2 mm CaCl2 and preincubated with 15dPGJ2 or vehicle control (0.1% v/v DMSO). Clot retraction was initiated by adding an equal volume of 2 U mL−1 thrombin and left for 1 h at room temperature before the weight of the clot was measured.Human washed platelets at 5 × 10m) was studied in vitro using microfluidic flow cells  as described previously m), was perfused through collagen‐coated (100 μg mL−1) Vena8Biochips at a shear rate of 20 dyn cm−2. Platelet adhesion was determined by comparing fluorescence intensity in the vehicle and treated samples.Adhesion on collagen in the presence of integrillin (10 μ−1) for 5 min under non‐stirring conditions, washed platelets (4 × 108 cells mL−1) were lysed in an equal volume of NP40 buffer  NP‐40, pH 7.3) and proteins of interest isolated by immunoprecipitation using 1 μg mL−1 of appropriate antibodies. Prior to immunoblotting, which was performed as described previously Following adhesion to fibrinogen or stimulation with thrombin . Values obtained in several experiments were converted into percentages for comparison of controls with treated samples. P ≤ 0.05 was considered statistically significant. Unless stated otherwise, values are expressed as mean ± SEM; n values are ≥ 3.Statistical analyses were performed on data using GraphPad prism software . Data were analyzed using Student's −1) were analyzed in the presence and absence of PPARγ receptor agonists. Additional platelet agonists were not added, to enable the study to focus (primarily) on outside‐in signaling through αIIbβ3. As shown in Fig. m) caused ~ 50% inhibition of adhesion and a significant reduction in spreading. A reduction in surface area coverage was observed following treatment with increasing concentrations of 15dPGJ2 (data not shown) and ~50% fewer platelets generated lamellipodia compared with vehicle control (0.1% v/v DMSO). Inhibition of both adhesion and spreading was also observed in the presence of inhibitors of ADP and thromboxane A2 , both thiazolidinediones, also caused inhibition of both adhesion and spreading on fibrinogen compared with the untreated control  did not alter platelet adhesion or spreading on fibrinogen under static conditions compared with vehicle control  caused a significant reduction in myosin light chain phosphorylation compared with vehicle control (0.1% v/v DMSO) in both thrombin (0.1 U mL−1)‐stimulated and fibrinogen (100 μg mL−1)‐adhered platelets   Figure A. This s−1)‐adhered platelets. The number of adhered platelets was normalized to ensure any alterations in phosphorylation were not a result of altered adhesion. Adhesion to fibrinogen was associated with an increase in phosphorylation of β3 and, as shown in Fig. m 15dPGJ2 or ciglitazone caused a reduction of ~50% and ~30%, respectively, in β3 Y773 phosphorylation in comparison with vehicle control‐treated platelets.Phosphorylation of integrin β3 at Y773 (Y747 in mice) is essential for outside‐in signaling by αIIbβ3 This reduction in β3 phosphorylation may alter the ability of αIIbβ3 to bind to fibrinogen and propagate outside‐in signaling, as previously published data show that PPARγ agonists reduce fibrinogen binding in agonist‐stimulated platelets m 15dPGJ2 in comparison with vehicle‐treated controls. These observations further support a negative role for the PPARγ agonists in the regulation of αIIbβ3 outside‐in signaling.A key step in the second wave of cytoskeletal rearrangements following the release of ADP and secondary mediators, is the phosphorylation and activation of focal adhesion kinase (FAK) and PI3‐kinases, which are essential for platelet spreading Negative regulators of platelet activation can function either by reducing positive signaling or by increasing inhibitory signaling. The cyclic nucleotides cAMP and cGMP, through activation of adenylyl cyclase and cyclic AMP‐dependent protein kinase (PKA) and soluble guanylyl cyclase and cyclic GMP‐dependent protein kinase (PKG), respectively, have both been shown to inhibit adhesion and spreading on fibrinogen and platelet cytoskeletal rearrangements m) for 10 min prior to lysis (resting samples) or adhesion to fibrinogen (100 μg mL−1). The samples were then analyzed for VASP phosphorylation at Ser239, which is the PKG selective phosphorylation site. Interestingly, although no alteration of VASP S239 phosphorylation was observed in resting platelets  platelets  at a concentration that was capable of reversing NO donor PAPA‐Nonoate (100 μm) mediated increases in VASP S239, thereby suggesting the increase in VASP S239 phosphorylation is independent of PKG activity  in VASP S157 phosphorylation in both resting and fibrinogen‐adhered platelets compared with vehicle alone. This increase in VASP S157 phosphorylation was also observed in ciglitazone‐treated platelets  or Rp‐8‐CPT‐cAMPs (100 μm) reduced both 15dPGJ2 and ciglitazone‐mediated increases in VASP S157 phosphorylation in resting and fibrinogen‐adhered platelets  did not prevent the 15dPGJ2 and ciglitazone‐induced increases in VASP S157 phosphorylation.  or AKT inhibitor AKT inhibitor IV (5 μm)  Figure , which pm) and ciglitazone ‐treated resting platelets in the absence and presence of a DP/EP receptor antagonist AH6809 (10 μm) m) Activation of PKA is widely known to occur following stimulation of Gs coupled receptors, including the prostaglandin receptors: the IP, DP and EP receptors Interestingly, and in further support of lack of involvement of Gs coupled signaling, no apparent alteration in cAMP levels was observed in resting platelets treated with increasing concentrations of 15dPGJ2 Fig. A and B. −1) compared with resting platelets and this interaction was significantly reduced following treatment with either 15dPGJ2 or ciglitazone  is a region that could undergo conformational changes, potentially altering its interaction with its binding partners, including β3 03 is a rThis study provides further evidence for a role for agonists of nuclear receptors in the negative regulation of platelet activation and supports previous reports that suggest PPARγ agonists are cardio‐protective. PPARγ agonists, thiazolidinediones, are currently used in the treatment of diabetes mellitus type 2. As there is an increased risk of cardiovascular disease associated with diabetes, treatment with PPARγ agonists could complement the effects of other antiplatelet therapies in reducing the risk of thrombosis.A. J. Unsworth designed the research, performed experiments, analyzed results and wrote the manuscript. N. Kriek and A. P. Bye performed experiments, analyzed results and edited the manuscript. K. Naran performed experiments and analyzed results. T. Sage and G. D. Flora performed experiments. J. M. Gibbins designed the research and wrote the manuscript.The authors state that they have no conflict of interest.Fig. S1. The kinetics of adhesion and spreading on fibrinogen.Fig. S2. PPARγ‐dependent inhibition of outside‐in signaling.Fig. S3. Ciglitazone inhibition of αIIbβ3 outside‐in signaling is associated with up‐regulation of PKA activity.Fig. S4. PPARγ up‐regulation of VASP S157 phosphorylation is not due to activation of PKC.Fig. S5. PPARγ ligand up‐regulation of VASP S157 phosphorylation is not dependent on PI3K or AKT activity.Fig. S6. PPARγ ligand up‐regulation of VASP S157 phosphorylation is not dependent on DP, EP or IP receptor activation.Fig. S7. Negative regulation of αIIbβ3 outside‐in signaling by PPARγ agonists.Click here for additional data file.


PIN auxin efflux carrier family possesses three highly conserved putative mitogen‐activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well‐characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6. In contrast to AGC kinases, MAPK‐mediated phosphorylation of PIN1 at adjacent sites leads to a partial loss of the plasma membrane localisation of PIN1. MAPK‐mediated modulation of PIN trafficking may participate in environmental adjustment of plant growth.Plant growth flexibly adapts to environmental conditions, implying cross‐talk between environmental signalling and developmental regulation. Here, we show that the  ER, endoplasmic reticulumHL, hydrophilic loopMAPK, mitogen‐activated protein kinaseMKK, MAPK kinasePAT, polar auxin transportPM, plasma membranempk4 mutants are severely dwarfed The mitogen‐activated protein kinase (MAPK) phosphorylation cascades are conserved signalling modules in all eukaryotes, consisting of three classes of enzymes, which are activated in a sequential phosphorylation cascade Such functional diversity and complexity of MAPK pathways imply complex substrate repertoires. Accordingly, human MAPKs phosphorylate a high number of functionally diverse substrates Plasma‐membrane‐localised PIN proteins are important in the establishment of polar auxin transport (PAT), a process generating auxin gradients, which are key determinants of organ formation and development. Plasma membrane (PM) localisation is generally associated with the presence of a long hydrophilic loop (HL) (PIN1/2/3/4/7), while short‐HL PINs (PIN5/8) are predominantly localised to the endoplasmic reticulum (ER) Auxin transporters are subject to post‐translational modification, and the characteristic polar membrane localisation of PIN is regulated by phosphorylation of PIN by the protein kinase PINOID (PID) and the related WAG1/2 kinases In this work, we set out to investigate three uncharacterised conserved MAPK phosphorylation sites in long‐HL PIN proteins adjacent to the similarly conserved PID sites. As both of these phosphosites can be traced back to the common ancestor of land plants, they might play a connected role in the regulation of PM‐localised PINs.pGEM‐T Easy vector . The c‐myc epitope tag was introduced to generate N‐terminal epitope‐tagged derivatives. To generate a plant transformation vector the myc:MKK7 fragment was first subcloned into the Gateway entry vector pENTR4 . From pENTR4, the tagged gene version was moved into the expression vector pER8GW, a Gateway version of the β‐estradiol‐inducible expression vector pER8 pER8GW:myc:MKK7 (pER8:MKK7).MKK7 was amplified from genomic DNA (intronless gene) and was cloned into PIN1 cDNA was amplified and cloned into pGEM‐T Easy vector (Promega). For in vitro transcription/translation the HL sequence was subcloned into pEU3‐NII‐GLICNot vector by ligation independent cloning 35S:gPIN1:GFPThe hydrophilic loop (HL: residues 156–482) of Site‐directed mutagenesis reactions were performed using QuikChange Lightning Site‐Directed Mutagenesis Kit . Nonphosphorylatable  and phosphomimetic  PIN1 versions were generated by sequential mutagenesis reactions using the following primers: PIN1‐T248A: F: GTTCAAGAAACCCAGCGCCGCGGGGCTCTAGTTTTAATC, R: GATTAAAACTAGAGCCCCGCGGCGCTGGGTTTCTTGAAC; PIN‐T286A: F: GGTTCTAAAGGTCCTGCTCCGCGGCCTTCCAACTACG, R: CGTAGTTGGAAGGCCGCGGAGCAGGACCTTTAGAACC; PIN‐T226A: F: CGAGAAGGTCTCAAGGCTTAAGCGCTGCACCTAGACCTTCGAATC, R: GATTCGAAGGTCTAGGTGCAGCGCTTAAGCCTTGAGACCTTCTCG; PIN‐T248E: F: GAGTTCAAGAAACCCAGAGCCTAGGGGCTCTAGTTTTAATCATAC, R: GTATGATTAAAACTAGAGCCCCTAGGCTCTGGGTTTCTTGAACTC; PIN‐T286E: F: CTGTGTTTGGTTCTAAAGGTCCTGAGCCTAGGCCTTCCAACTACG, R: CGTAGTTGGAAGGCCTAGGCTCAGGACCTTTAGAACCAAACACAG; PIN‐T226E: F: CGAGAAGGTCTCAAGGCTTATCTGCAGAACCTAGACCTTCG, R: CGAAGGTCTAGGTTCTGCAGATAAGCCTTGAGACCTTCTCG. Mutant clones were verified by sequencing.Arabidopsis thaliana Col‐0 was used as genetic background. The T‐DNA insertion line Salk_073907 for mpk6 was obtained from the Nottingham Arabidopsis Stock Centre. The ProPIN1:PIN1:GFP transgenic line was published in Ref. −2·s−1 cool white light under long day (16 h of light/8 h of dark) conditions. Transgenic Arabidopsis lines were generated using the floral dipping method in vitro mRNA synthesis was carried out using TranscriptAid T7 High Yield Transcription Kit  according to the manufacturer's instructions. Cell‐free translation was carried out by using WEPRO7240H Expression Kit . In order to activate His‐tagged MPK4 and MPK6 when included in the phosphorylation assay mix, mRNA encoding a constitutively active myc:MKK1 and myc:MKK4, respectively, were also added to the translation mixture as described In vitro‐translated His6‐AtMPK4 and His6‐AtMPK6 proteins were purified by affinity chromatography on TALON Magnetic Beads (Clontech), in vitro‐translated GST‐PIN1loop and GST‐PIN1loop‐3A were purified by affinity chromatography on Glutathione Magnetic Beads (Thermo Scientific) The in vitro‐translated, affinity‐purified substrate and kinase were used respectively. As an activity control, 10 μg myelin basic protein was used as a generic MAPK substrate (not shown). The assays were carried out in 20 mm HEPES, pH 7.5, 100 μm ATP, 1 mm DTT, 15 mm MgCl2, 5 mm EGTA and 5 μCi [γ‐32P]ATP with bead‐bound GST‐PIN1loop or GST‐PIN1loop‐3A as substrates for 30 min at room temperature, and then stopped by the addition of Laemmli SDS buffer. Samples were fractionated by SDS/PAGE. The gel was fixed, stained with Coomassie Blue, dried and analysed by autoradiography. The kinase assay was performed three times with similar results.For kinase assays, 300 and 100 ng of In vitro phosphorylation reaction mixtures were separated on NuPAGE 4–12% Bis‐Tris gel (Thermo), bands corresponding to the GST:PIN1HL protein were excised from the gel and subjected to in‐gel digestion using side chain‐protected porcine trypsin (https://msf.ucsf.edu/protocols.html). Approximately 75% of the peptide mixtures were subjected to phosphopeptide enrichment by IMAC using Fe‐nitrilotriacetic acid proteome discoverer software  were searched against the Swissprot database  using the ProteinProspector search engine (v.5.18.0.). Search parameters: enzyme – trypsin with maximum 1 missed cleavage per peptide; fixed modification – carbamidomethyl (Cys); variable modifications – acetylation (protein N terminus), oxidation (Met), pyroglutamic acid formation  allowing maximum two variable modifications per peptide; mass accuracy – 5 p.p.m. for precursor ions and 10 p.p.m. or 0.6 Da for fragment ions . Subsequently, another search was conducted on the subset of confidently identified proteins using the same search parameters except that a maximum of two missed cleavage sites per peptide were allowed, and phosphorylation on Ser/Thr/Tyr was also set as variable modification allowing a maximum of three variable modifications per peptide. For all searches the following acceptance criteria were applied: score > 22 and 15, and E‐value < 0.01 and 0.05 for protein and peptide identifications respectively. For phosphopeptide site assignments, SLIP threshold pER8:MKK7 was achieved by treatment with β‐estradiol for 4 h. flg22 treatment was carried by the application of 4 μm custom‐synthesised flg22 peptide for 12 h. RFP‐fused organelle markers are described in Ref. Protoplasts were prepared from an Arabidopsis wild‐type Col‐0 root suspension culture and transiently transformed with 5 μg of each plasmid constructs as described Samples were fixed and processed as described in Ref. Confocal laser scanning microscopy analysis of PIN1:GFP samples was performed using a Leica TCS SP8 confocal microscope  equipped with a HC PL APO 63.0 × 1.4 OIL CS2 objective, and a GaAsP detector. Excitation/emission wavelengths of GFP and organelle‐specific RFP markers were 488/505–550 and 561/565–620 nm respectively.For protein gel blotting, equal protein amounts from plant extracts were separated by SDS/PAGE, transferred to polyvinylidene difluoride membranes , and probed either with horseradish peroxidase‐conjugated anti‐myc monoclonal antibody  or anti‐HA (Roche) and anti‐GFP  primary antibodies, detected by HRP‐conjucated secondary antibody (GenScript). The membranes were visualised with enhanced chemiluminescence substrate (Thermo Fisher Scientific) and exposed on CL‐Xposure film (Thermo Fisher Scientific).In silico analysis of Arabidopsis PIN protein sequences by using the Eukaryotic Linear Motif Resource (ELM) Physcomitrella) lineages  are conserved to the same degree and are consistently located in the close proximity (i.e. four residues downstream) of the conserved putative MAPK sites. As AGC kinases do not require an upstream TP motif There are two additional MAPK phosphorylation sites further downstream in PIN1 HL (S337 and S377), which in contrast do not possess the flanking preference motifs and PID phosphorylation site. S337, however, was found to be a MPK6 target in planta phosphorylation suggest functional importance of T227, T248 and T286, therefore we tested their MAPK‐mediated phosphorylation. To this end, in vitro kinase assays were performed first  resulted in a marked diminution of phosphorylation. These results indicate that T227, T248 and T286 are MAPK phosphorylation sites on PIN1. To directly demonstrate the MPK6‐mediated phosphorylation of these residues we performed LC‐MS/MS analyses of the tryptic digests of the MPK6‐treated and control GST‐PIN1 HL. Phosphorylation of T227, T248, T286 and the previously reported S337 Wide‐ranging conservation of MAPK phosphorylation site patterns and their co‐occurrence with the PID sites along with proven Physcomitrella long‐HL PIN sequences implies a very early formation of the MAPK‐PIN regulatory link during land plant evolution. Separation of the group A and B MAPK clades most probably took place after the separation of the flowering plant lineage from more ancient lineages such as mosses and lycophytes, possibly as an adaptation to the formation of complex body architectures The conservation of the MAPK phosphorylation sites corresponding to T227, T248 and T286 of PIN1 down to the bud1 mutant inhibits PAT and leads to developmental anomalies and the MKK7‐MPK6 module has been reported to modulate PIN1 polarity in the xylem parenchyma cells of the basal inflorescence stems MKK7, which was shown to activate downstream MAPKs Moderate overexpression of MKK7 in the MKK7 transgene not treated with β‐estradiol also presented a normal PIN1 localisation pattern  localised even when it is overexpressed, and therefore this experimental system was previously utilised to study the regulation of PIN1 PM localisation ots Fig. A. Represots Fig. . In protots Fig. B, implyiExpression of a phosphomimetic mutant version of the sites in question on PIN1:GFP T227E/T248E/T286E) resulted in alterations of PIN1:GFP subcellular localisation similar to that observed in response to MPK6 activation, but in an exaggerated form: aggregates of extremely large size were commonly formed  group. Conservation of ‘double preference’ residues may be plausibly interpreted as indirect evidence of the coevolution of two phosphorylation sites through a motif merger. Thus, functionality of MAPK‐mediated phosphorylation may be at least as complex as in the case of PID, requiring further studies to dissect details of this novel regulatory mechanism.It was recently reported that PIN1 is also phosphorylated by MPK6 at S337 located C‐terminally at a distinct region and the phosphorylation status of this residue apparently participates in the regulation of PIN1 polarity in a specific developmental setting of xylem parenchyma cells in 35‐day‐old basal inflorescence stems mpk6 mutant we also demonstrate that this is an MPK6‐mediated process. Considering that flg22 activates further MAPKs besides MPK6, including MPK4, this result was rather unexpected. A plausible explanation is differential accessibility of PIN1 through kinase localisation . Expression and cellular localisation of both MPK6 and MPK4 in root cells have been studied in detail. While MPK6 is localised to the trans‐Golgi network and the plasma membrane in planta under specific conditions cannot be ruled out entirely. A flagellin‐induced delocalisation of PIN1 is also in line with the severe retardation of root growth in response to flagellin treatment Polar auxin transport and the resulting local auxin maxima sites are important in establishing developmental patterns in plants. PINs determine the direction of PAT through their asymmetric subcellular localisation and thus signalling pathways regulating PIN localisation can modulate developmental programs in response to triggering stimuli The MAPK pathways are central signal transduction pathways in all eukaryotes. In both mammals and flowering plants they regulate a plethora of processes. Understanding the mechanisms of such complexity requires a detailed dissection of MAPK networks, yet our knowledge on MAPK substrate phosphorylation in plants is limited. Our results provide evidence for the co‐option of MAPKs together with AGC kinases at an early stage of land plant evolution to regulate PM‐localised PIN targeting, in line with the challenges of terrestrial lifestyle. Environmentally activated MAPK pathways constitute a novel regulatory mechanism of PIN, which may play a role in the environmental plasticity of plant development.Fig. S1. Sequence alignment of Arabidopsis, rice and Physcomitrella patens members of the UNIPROT PIN auxin efflux protein family.Fig. S2. Spectra for representative identified phosphopeptides derived from PIN1 hydrophilic loop.Fig. S3. MAPK phosphorylation status at T227, T248 and T286 influences intracellular trafficking of PIN1.Click here for additional data file.


Feral swine are globally known as one of the most destructive invasive vertebrates, damaging native habitats, native plants and animals, agriculture, infrastructure, spreading diseases. There has been little quantification on their disturbance to archaeological sites across a broad landscape. Over 6 years we inspected 293 significant archaeological sites for swine disturbance across a vast area. We found a 42% prevalence of swine disturbance among all sites, with prevalence not distinguishable among prehistoric sites, historic sites, and sites with both components. The areas of disturbance mapped within three historic homestead sites showed 5–26% of total site surface area rooted. Disturbance was not evident upon re-inspection of one of these sites after 18 months, indicating how evidence of disturbance can be obscured in this environment. Thus, our observed 42% prevalence of disturbance should be considered a minimum for disturbance occurring through time. Artifacts depths were <10 cm of the surface at 85% of the sites and <20 cm of the surface for 90% of the sites. Feral swine rooting commonly exceeds 20 cm in depth, especially in soft sandy substrates typical of Florida, making the great majority of the studied sites highly vulnerable to artifact damage or displacement. Sus scrofa) to North America in Florida, and many additional introductions, both intentional and accidental, have followed124678410In 1539 the Spanish explorer de Soto first introduced feral swine  is the largest Air Force base in the USA and approaches the size of the smallest state in the U.S., Rhode Island. Approximately 86% of Eglin AFB is forested, 12% is dedicated solely to military activities , and the remaining 2% is water, marshes and barrier islandThe base’s stewardship obligation towards cultural sites is addressed in part by an active site monitoring program that checks site conditions throughout the year to safeguard sites when they may be impacted by a variety of land use threats, including military training activities, impacts from forestry management such as prescribed burns, and recreational use. This monitoring program was augmented in 2010 to include documentation on whether feral swine disturbance was present within each visited site, and from 2010–2016, 293 of the base’s 579 significant archaeological sites were examined for feral swine disturbance. Detailed spatial assessment of disturbance was included for three historic sites where the areas of disturbance were mapped by GPS. We report here on the results of these surveys and the implications for protecting archaeological sites in areas where feral swine occur.2 = 3.13, df = 2, p = 0.21), with disturbance at 38% of prehistoric sites, 49% of historic sites and 38% of sites with both prehistoric and historic components , 103 historic sites (35%), and 86 sites having both prehistoric and historic components (29%). Across all sites, feral swine disturbance was observed within 122 (42%). The prevalence of swine disturbance did not vary extensively among the broad site age classifications . Of the 261 sites with artifacts within 20 cm of the surface 113 (43%) had feral swine disturbance, not distinguishable from the prevalence (30%) for sites without artifacts within 20 cm of the surface .The archaeological records originally establishing site significance included information on artifact depth and characterizations of their landscapes and vegetation. We were particularly interested in identifying those sites most susceptible to swine disturbance based on artifact depth. Swine are known to root as deep as 1 m12192, of which the area of disturbance within the site boundary totaled 774 m2, or 15% of the total site area (2 (5%) of the total 12716 m2 of the site. Over 1100 artifacts were recovered from this site in previous archaeological site assessments, with materials recovered as deep as 40 CMBS, but most were in the upper 20 cm of deposits, also making this site highly vulnerable to disturbance. In a follow-up visit in February 2016, feral swine disturbance was not apparent due to the weathering processes on the soft, sandy soil substrate. At site 8WL1471, a homestead dating to 1904, six separate areas where the ground surface was heavily disturbed by swine were identified and measured, . A total of 195 artifacts were recovered in previous archaeological site assessments between 0–60 CMBS, a majority of which were recovered from the upper 40 cm. Due to the large amount of materials on or near the surface, this site also was highly vulnerable to swine disturbance. This site was also visited March 2013 and March 2016, with feral swine disturbance observed, but not mapped on those occasions.The 8OK163 site, a homestead claim dating to 1905, was found to have 4 patches of feral swine disturbance within its perimeter. The total site area covered 5186 mite area . Culturaite area . The comeasured, . The comEach of these three sites were visited 3–7 times from 2010–2016 as part of safeguard checks on archaeological sites during base activities that might threaten them. Quercus spp) found at these three sites. This attractant for feral swine rooting could affect disturbance at over half of the archaeological sites on the base, because over half (52%) are characterized with oak trees within the site.Rooting prominently seen during the three September 2014 mapping sessions was likely due at least in part to feral swine rooting for a mast crop produced by abundant oak trees . Sites were visited on the basis of need to safeguard them against planned base activities that could negatively impact them, with prime examples being military training activities, forestry management including prescribed burns, and recreational uses. Such safeguard monitoring is part of base protocol to fulfill required stewardship legal obligations as set forth by the Archaeological Resource Protection Act and the National Historic Preservation Act. Beginning in June 2010 to February 2016 we also included inspection for feral swine disturbance as part of the site monitoring program, with swine disturbance identified as ground overturned from rooting activityPrevious archaeological surveys had categorized sites according to whether they were prehistoric, historic, or had both, and data on depths at which artifacts could be found were also available. We used the artifact depth data to assess vulnerability of sites to swine rooting, paying particular attention to sites with artifacts known to occur within 10 cm of the surface, and those with artifacts known to occur within 20 cm of the surface.Comparison of prevalence of swine disturbance among site age categories  was conducted using a chi-square contingency table test. Similarly, comparison of disturbance prevalence among sites with and without artifacts within 10 cm of the surface and comparison of disturbance prevalence among sites with and without artifacts within 20 cm of the surface were also conducted using chi-square contingency table test.th century.For a more thorough investigation of within-site disturbance areas, we evaluated the spatial extent of swine disturbance across the geographical area of three historic homesteads. A Global Positioning System (GPS) was used to overlay a series of transects across the site area. These predetermined transects were walked and the beginning and end points of disturbed areas were marked with flagging tape. This effort clearly defined the perimeters of disturbance across a location, which were then recorded using GPS. The recorded shapes were imported into a Geographic Information System (GIS) to calculate areas of the resulting polygons. Of the three sites selected for this more intensive documentation of the feral swine disturbance, two were within Okaloosa County and the third was in Walton County. All three sites represented homesteads that date to the early 20Site 8OK163 was originally identified in the 1980’s during the Eglin Historic Preservation Plan and then tested in 1998. The site likely represents the homestead claim of Horace Jones dating to 1905. This historic site is situated in level uplands at an elevation between 58 and 59.5 m above mean sea level (AMSL). Bull Creek is the closest water source and is 350 m to the east. Vegetation is slash pine forest with mixed hardwoods, with a number of small oaks noted within the site boundary.The 1998 investigation consisted of a surface search, the excavation of 18 50 cm × 50 cm shovel tests and a metal detector sweep. One large brick concentration was identified on the surface and appeared to be the location of a structure. The investigation recovered 86 artifacts as deep as 40 CMBS, with the majority in the upper 20 cm of deposits. These artifacts indicated a 1900 to 1920 occupation date. Mr. Jones sold the property to the Union Naval Store which operated a turpentine still just to the east of this site. Site 8OK163 was determined to be eligible for listing on the National Register of Historic Places due to the unique opportunity this site presented in comparative analysis with a larger commercial enterprise of the same time frame. From June of 2010 to February of 2016, Site 8OK163 was visited on seven occasions. In September 2014, the.feral swine disturbance was mapped to document the extent of the disturbance.Site 8OK1062 was initially discovered in 1997 and was further tested and determined eligible for listing on the National Register of Historic Places in 2005. This site, the 1905 homestead of James F. Edge, is situated on a ridge-nose that is bound to the north by the Tenmile Creek floodplain and the west by an unnamed tributary of Tenmile Creek. The site is on level uplands at the elevation of 15 m AMSL. The landform slopes sharply towards the west into an unnamed tributary of Linton Spring Branch. Vegetation is primarily sand pine forest, with live oak, laurel oak, magnolia and persimmon scattered across the site.The larger of two brick concentrations seem to represent the main house while the smaller brick concentration may be a smoke house. Over 1100 artifacts were recovered from this site, with much of this material recovered from the upper 20 cm of soil. Site 8OK1062 was visited for this study on four occasions from June of 2010 to February of 2016. Feral swine disturbance was mapped during September 2014.th century which may be associated with the 1904 homestead claim of James Linton. Due to the relatively high artifact density, variation in horizontal surface distribution of materials and the association with a known land claim, site 8WL1471 was listed as potentially eligible for listing on the National Register of Historic Places and must be protected until further investigations can be conducted. Site 8WL1471 was visited on three occasions during this study and was mapped during the visit of September 2014.Site 8WL1471 was initially identified in 1998 during an archaeological survey along the waters of Linton Spring Branch. The site was identified when a scatter of historic material was noted on the exposed ground surface on either side of an abandoned road. At that time researchers carried out a surface search and the excavation of 26 50 cm × 50 cm shovel tests. During this investigation three brick concentrations were noted as well as a number of small depressions that were believed to represent cultural features such as a well, trash pits or privy. A total of 195 artifacts were recovered from surface and subsurface context between 0–60 CMBS, with a majority of the remains being recovered from the upper 40 cm. The artifacts indicated the site represented a homestead dating to the early 20How to cite this article: Engeman, R. M. et al. Prevalence of feral swine disturbance at important archaeological sites over a large landscape in Florida. Sci. Rep.7, 40287; doi: 10.1038/srep40287 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Topographical material surface features are sensed by cells and provoke a large range of cellular responses. We recognized earlier, that at micropillar topographies in the range of 5 µm, the osteoblasts attempt to phagocytize the pillars resulted in increased energy requirements and reduced osteoblast marker expression, e.g., collagen type I and osteocalcin. However, the precise cellular signaling transducing the topographic information into the cell and evoking phagocytic processes remained unknown. Here, we could show that the RhoA/ROCK signaling is involved in the transduction of the topography-mediated cellular reactions. After inhibition of ROCK-2 with Y27632 for 24 h, no caveolae-mediated micropillar assembly of the cell membrane domain component caveolin-1 (Cav-1) was found. ROCK inhibition was also able to attenuate the pillar-induced decrease in β-actin. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibition with LY294002 for 24 h did not influence the Cav-1 clustering on micropillars. Our results illustrate the importance of the integrin down-stream signaling of RhoA/ROCK in the recognition of and adaption to surface microtopographies by osteoblasts and extend our understanding about the complex mechanism of action inside the cells. In cell-biomaterial interaction, knowledge of the dependence of cell behavior on topographical features is relevant for the design of implant surfaces. Cells are able to recognize surface topographies of micron as well as nanometer size and adapt their cellular behavior by sensing topographies down to 10 nm with their filopodia . Micron-Integrin-dependent functions can be altered by cellular morphology changes and can modulate the integrin-activated signaling mediators. Rho-family GTPases and their downstream kinase Rho-associated kinase (ROCK) relay integrin-derived signals; they also organize the actin cytoskeleton. Therefore, it is suggested that they integrate cell shape and function . RecentlIn this study, we examined the cell signaling in human MG-63 osteoblasts depending on the underlying micropillar topography and the topography-triggered cell changes. The artificial micropillar topography has the advantage of constant repetitive dimensions and facilitates the specific analysis of topography-induced cellular processes; it also highlighted the importance of the cell-material contact area for the osteoblasts in maintaining their characteristic osteoblast function  and showPeriodically microtextured samples (size 1 cm²) with regular cubic pillar geometry on the surface having a dimension of 5 × 5 × 5 µm in width x length x height and 5 µm in spacing (P-5 × 5) were used. Unstructured, planar silicon wafers (Ref) were employed as controls. The samples were fabricated by deep reactive-ion etching (DRIE)  on silicon wafers and coated with an additional 100 nm titanium (Ti) layer, as reported before , 10.The human osteoblast-like cells MG-63  were cultivated in Dulbecco’s modified eagle medium  with 10% fetal calf serum (FCS) , as was reported before , 10. ForImmunoblots were performed from total lysates of MG-63 cells which were cultivated on the Ti arrays. The BioPlex cell lysis kit  was used. Protein quantification was performed using the Bradford method (Bio-Rad Laboratories GmbH). Total cellular protein was separated by SDS-PAGE (Bio-Rad Laboratories GmbH) and afterwards transblotted to polyvinylidine fluoride (PVDF) membranes . Analyses were done with the following antibodies: β-actin mouse monoclonal , caveolin-1 rabbit polyclonal , Tyr14 phosphorylated caveolin-1 rabbit monoclonal , FAK mouse polyclonal (BD Transduction), ROCK-2 pY256 rabbit monoclonal , ROCK-2 rabbit polyclonal (Santa Cruz Biotechnologies Inc.), RPLP0  mouse monoclonal , and Src mouse monoclonal (New England Biolabs GmbH). The membranes were incubated with the appropriate primary antibody over-night at 4°C followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (New England Biolabs GmbH). Primary antibody binding was detected by using a fast chemiluminescent substrate for HRP . For each protein detected, at least four independent experiments were performed. Immunoblotting analyses were carried out with ImageLab-ChemiDoc-MP (Bio-Rad Laboratories GmbH) and the densitometric analysis with ImageJ . The 60S acidic ribosomal protein P0 (RPLP0) as well as the stain-free gel with total protein loading were used as an endogenous control. For the evaluation of the phosphorylation states, the normalized phosphorylated protein was calculated relative to the normalized unphosphorylated, total protein.Total lysates of MG-63 cells were prepared with the BioPlex cell lysis kit (Bio-Rad Laboratories GmbH) and protein quantification was performed using the Bradford method (Bio-Rad Laboratories GmbH), just as for the immunoblots. For the luminex assay measurements, the following analytes and kits were used: for the focal adhesion kinase (FAK) (pY397) and FAK (pY861) the MILLIPLEX MAP Kit , and for Src (pY419) the MILLIPLEX MAP Human Src Family Kinase Kit (Merck KGAA). Protocols were performed according to the manufacturer’s instruction in a 96-well plate with 25 µl of the protein lysates for cellular protein quantification. Protein quantification was done using the Bio-Plex-System (Bio-Rad Laboratories GmbH) and the Bio-Plex Manager™ 4.1.1 software (Bio-Rad Laboratories GmbH), measured in mean fluorescence intensity (MFI). The MFI values were normalized using protein lysate concentrations measured according to the Bradford method. For the evaluation of the phosphorylation states, the normalized phosphorylated protein was calculated relative to the normalized unphosphorylated, total protein.Immunofluorescence staining was performed as reported before , 10. Brin = 50 cells per sample out of n = 2 experiments with ImageJ software  using the LSM images from the actin cytoskeleton staining.Image acquisition was done on an inverted confocal laser scanning microscope LSM 780 (Carl Zeiss AG) using the ZEISS oil immersion objective (C-Apochromat63) and the ZEN 2011 (black version) software (Carl Zeiss AG). All images were displayed as three-dimensional (3D) z-stacks . Z-stacking was used to generate a 3D representation to understand the overall cell structures. Thus, a false interpretation due to different observation levels  could be avoided. The cell areas for P-values <0.05 were considered to indicate significant differences.Statistical analyses were carried out with GraphPad Prism5 software (GraphPad Software Inc.). Results are presented in box plots with medians, quartiles and an interquartile range (IQR) ± 1.5 x IQR. Data analyses were performed using the Mann–Whitney U test. The overall cell interaction with the microtopography is illustrated in Fig. The protein amount of paxillin, talin and vinculin as well as the phosphorylation state of paxillin were unchanged in MG-63 cells after 24 h on the micropillars (data not shown).The development of focal adhesions and the regulation of actin stress fiber formation are stimulated by the small GTPase RhoA . The majThe integrin downstream signaling analysis showed alteration in the Rho/ROCK pathway for cells on the micropillars. Therefore, the cells were treated with the ROCK inhibitor Y27632. Rho/ROCK signaling is associated with alterations in the properties and signaling of membrane protrusions  and has The Cav-1 clustering on pillars was alleviated due to ROCK inhibition with Y27632. We wanted to know, if the protein expression level was also altered. Western blot analysis for the pCav-1 relative to the total Cav-1, showed a significantly decreased level after treatment with Y27632, but independent on the surface topography. This general lowering of the pCav/Cav levels results from a minor increase in Cav-1 protein expression after ROCK inhibition. After all, the micropillar-induced trend for an enhanced pCav/Cav was also observable after ROCK inhibition Fig. . The pro2) and phosphatidylinositol-3,4,5-trisphosphate P3), displayed a changed localization in the cell membrane of MG-63 cells on the micropillars towards the top and around the micropillars after 24 h  signaling pathway plays a central role in the regulation of cell signaling, survival and proliferation. However, PI3K phosphorylation as well as the protein amount of PI3K showed no changes in MG-63 cells grown for 24 h on the micropillars (data not shown). In contrast, the phosphatidylinositol lipids, including phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate PTo see the PI3K influence on the organization of cellular structures more in detail PI3K inhibition with LY294002 was performed. But we revealed no alterations in MG-63 cells on micropillars concerning the intracellular actin cytoskeleton and the caveolae performance after 24 h Fig. .Fig. 5CaOsteoblasts are attempting to internalize the micropillar surface structures via a caveolae-mediated phagocytosis, to possibly establish the highest cell-surface contact to maintain their osteoblast function , 10. TheIn our experiments with osteoblasts on micropillars (24 h) the following scaffolding and signaling molecules were expressed in a stable, unchanged manner: the protein amounts of paxillin, talin, vinculin, PI3K, Rac, RhoA as well as the phosphorylation states of paxillin, PI3K, extracellular-regulated kinase (pERK1/2/ERK1/2), and mitogen-activated protein kinase (p-p38 MAPK/p38 MAPK) (data not shown).However, the micropillar topography provides the MG-63 cells a reduced adhesion area; therefore they probably display less integrin-mediated autophosphorylation of FAK at its Y397. Because fewer binding sites at the FAK are accessible for the Src kinase, this results in the reduction of the FAK Y861 phosphorylation , 20. FurConsequently, the cells showed signaling enhanced for motile processes, such as the reported caveolae-mediated phagocytosis accompanied with an increased Cav-1 phosphorylation (pCav-1) . The pCa2 as a substrate and converts it into PIP3 [2 is located in nascent endosomes and is converted by the PI3K to PIP3 in the late engulfment phase. Thus, the PI3K is essential for the closure of the endocytic vesicle [Phosphatidylinositols also play a central role in endocytic processes, such as the reported caveolae-mediated phagocytosis . These p3,4,5)P3 . During  vesicle . But on  vesicle .After ROCK inhibition, the cells may establish the highest cell-surface contact while avoiding the caveolae-mediated micropillar phagocytosis by performing the high branched morphology. The ROCK inhibition did not alter Cav-1 phosphorylation, which elucidates the importance of ROCK in the Cav-1 activation for cluster formation and verifies that ROCK is an important component for the topography-sensing and processing of the osteoblasts. In the later phase of adhesion, RhoA/ROCK are activated for transmission of tension and integrin ligation , which aThis study highlights the involvement of the intracellular signaling protein ROCK concerning the recognition of surface topographies by osteoblasts as well as in the transduction of the topography information in cellular responses. In detail, we could show that the inhibition of ROCK-2 with Y27632 impairs cellular processes such as phosphorylation of caveolin-1 and actin clustering around sharp-edged microtopographies, which are involved both in the attempt to phagocytize the micropillars. The phosphatidylinositol 3-kinase (PI3K) inhibition with LY294002 for 24 h did not influence the Cav-1 clustering on micropillars. The understanding of cellular signaling in more detail is important to find out specific cell parameters which react sensitive to topographic features of the biomaterial surface.


Aim. Transpedicular screw fixation is widely used in spinal surgery. But the insertion of pedicle screws can sometimes be challenging because of the variability in pedicle size and the proximity of nerve roots. Methods. We detected intraoperatively the sensitivity for iatrogenic pedicel perforation with a hand-held electronic conductivity measurement device (ECD) that measures electrical conductivity of tissue-medium surrounding the instrument tip. ECD was used to guide the placement of 84 pedicle screws in 15 patients undergoing surgery for tumor or degenerative spinal disease at various spinal levels from T8 to L5. Additionally a CT-scan controlled screw positioning postoperatively. Results. The placement was “correct”  for 78 of 84  screws, “suboptimal but acceptable” (0–2 mm penetration) for 4 of 84  screws, and “misplaced” (penetration > 2 mm) for 2 of 84  screws. Conclusion. Although this study was not designed to compare electronic conductivity technique to other guidance methods, such as fluoroscopy or navigation, a convincing “proof of concept” for ECD use in spinal instrumentation could be demonstrated. Advantages include easy handling without time-consuming setup and reduced X-ray exposure. However, further investigations are necessary to evaluate i.a. the economic aspects for this single-use developed instrument. Transpedicular screw placement in the vertebra during posterior operations to stabilize the spine is currently the most widely used and successful technique to treat pathological changes of the spine caused by trauma, tumor, scoliosis, or degenerative diseases –5. But cCurrently, various methods are used to attain the most correct pedicle screw placement possible. Among the mechanical aids, there is the possibility of probing the pedicle canal and inserting the pedicle screw by means of a guidewire , 12. AmoCurrently, an electrical impedance and conductivity measurement device was developed to improve the accuracy of pedicle screw placement. The probe-shaped tool can be used as a mechanical tool for the preparation of a lead canal for the insertion of the pedicle screw. It furthermore measures the electrical conductivity in the surrounding tissue, which changes depending on the tissue type, as shown in earlier animal and clinical feasibility studies –26. ThesThis paper reports on our initial experience with this device in severe degenerative disease and spinal tumor surgery.The electrical conductivity device PediGuard (ECD)  comes in three sizes (diameter × length in mm): 2.5 × 40, 3.2 × 45, and 4.0 × 44 , all of The ECD was used to place a total of 84 polyaxial screws into 15 patients. The procedures were performed between October 2008 and October 2011. There were 6 male and 9 female patients with a mean age of 61 years (41–83 y). Patients with cardiac pacemakers and severe osteoporosis were excluded from participation according to the manufacturer's recommendations. In 8/15 patients a degenerative disease with DDD, spinal canal stenosis, and spondylolisthesis was the indication for dorsal instrumentation with a screw/rod system. In the other 7/15 cases a metastatic tumor of the spine  was operated on. There were 7 single-level and 8 multilevel procedures .The instrument set was used for dorsal transpedicular stabilization operations on the thoracic and lumbar spine. The access to the vertebrae was in all cases via a dorsal midline access. The facet joints were prepared and an insertion canal was made with the ECD. Polyaxial screws  with a diameter of 5.5 mm (thoracic) or 6.5 mm (lumbar) and a length of 30–50 mm were placed into the vertebral bodies according to the trajectory given by the ECD. Postoperatively, CT imaging of the spine  was used to evaluate the positioning of the pedicle screw .The placement of the pedicle screws on postoperative CT was evaluated by an independent radiologist and graded into three levels: (a) correct, (b) suboptimal (but acceptable), or (c) misplaced . The posThirty-eight screws  were placed during posterior instrumented fusion (PLIF) for spondylolisthesis, while the other 46/84  were placed during dorsal instrumentation for spinal tumor .The signal remained constant, while the ECD was advanced forward through the pedicle into the vertebral body in 72/84 (85.7%) pedicle sites. In the other 12/84 (14.3%) sites, sound and LED signals warned for variation in the measured conductivity as a sign for possible pedicle wall penetration. In these cases, the ECD was slightly moved backwards and redirected in another trajectory through the pedicle until no further warning signal occurred.The screw placement was graded as “correct” for 78 of 84  screws and “suboptimal” for 4 of 84 screws . Hence, 97,6% of the screws were satisfactorily placed, whereas 2 of 84  screws had to be graded “misplaced” . In leveThe use of transpedicular screw systems during spinal surgery has become widespread , 5, 27, In this study, there was a high rate of correct placement of screws with the ECD  and a low rate of misplaced screws . This is consistent with previously published reports –25, as wThe present study has several important limitations that must be kept in mind. First, the study has no control group using other methods  as a basis for direct comparison. So we cannot draw any conclusions regarding improvement over conventional techniques. Thus, this study can only be regarded as a proof of concept for the electronic conductivity technique in spinal instrumentation. Second, the sample size is too small to serve as a conclusive evaluation, even if it had been designed as a randomized double-blind comparative study. Regrettably, it was not possible for us to enlarge the sample further. Third, the data were all from a single site; the rate of accuracy may be somewhat better or worse in the hands of other surgeons. Clinically, our initial experience was positive enough to warrant further investigation in carefully monitored research settings. We reemphasize the thought that this system has not yet undergone sufficient scientific evaluation for adoption into routine clinical practice . Such RCTs have to prove that the ECD technology really improves patient safety in direct comparison to other conventional methods of pedicle screw placement.Finally, we must comment on the cost of this device and associated design factors. The device cannot be reused for more than one operation—the outer casing of the device is made of a plastic, which makes it impossible to sterilize the instrument for reuse. The sound and light signals of the device are driven by a built-in internal battery that only lasts about 24 hours before dying out. In order to initially activate the battery, a paper-like tab on the side of the device must be pulled off. Once this paper-like tab is removed, the battery is active and running. There is no way to pause the battery from discharging. If the paper tab is accidentally removed before any operation is scheduled, the battery will die out anyway and the device would be useless. Furthermore, there is no way to open the device and replace the battery. Opening it up would require permanently breaking the plastic outer shell of the device, thus rendering it unusable. Moreover, the plastic shell has small airholes, so the user can hear the beeping tones from the speaker that is inside the handle. But consequently, blood or other patient fluids potentially carrying viruses can get inside the device through these sound-holes, thus contaminating the device so it cannot be safely reused on a second patient while the battery is still running. In the current era of dwindling healthcare budgets and limited resources for single-use instruments  one has to critically take this fact into consideration when deciding for such an “extra” tool. Even if this device later demonstrates a clinical benefit for patient health and safety (and not merely an increase in surrogate radiographic endpoints), rigorous health economic analyses are needed to determine whether it also has sufficient cost-benefit advantages. Potential ethnography endpoints for cost saving studies could include i.a. anesthetic case time and/or improved efficiency through lower instrument passes, leading to reduced overall operating time.The general engineering concept of measuring electrical conductivity to improve the accuracy of pedicle screw placement appears, in our small initial clinical experience, to be a potentially useful method. The ECD safely allowed detection of changes in the electromagnetic field around the instrument tip as a warning signal for tissue with different consistency to bone. With careful handling, it even allows detection of cortical breaches before full penetration has occurred, giving the surgeon the chance to redirect the trajectory. Further advantages of this technique include easy handling without a time-consuming setup and no additional X-ray exposure. However, further studies should evaluate the advantages of the system in cost-comparison and clinical benefit, because in our viewpoint the economic inefficiency of a single-use product, which is otherwise quite promising, may limit the use in routine spinal surgery.


The causes of the recent increase in Antarctic sea ice extent, characterised by large regional contrasts and decadal variations, remain unclear. In the Ross Sea, where such a sea ice increase is reported, 50% of the sea ice is produced within wind-sustained latent-heat polynyas. Combining information from marine diatom records and sea salt sodium and water isotope ice core records, we here document contrasting patterns in sea ice variations between coastal and open sea areas in Western Ross Sea over the current interglacial period. Since about 3600 years before present, an increase in the efficiency of regional latent-heat polynyas resulted in more coastal sea ice, while sea ice extent decreased overall. These past changes coincide with remarkable optima or minima in the abundances of penguins, silverfish and seal remains, confirming the high sensitivity of marine ecosystems to environmental and especially coastal sea ice conditions. Strong regional heterogeneity prevents thorough understanding of the recent increase in Antarctic sea ice. Here, analysis of marine and ice cores in the Western Ross Sea shows that late Holocene contrasting sea ice patterns between open and coastal areas are related to katabatic winds and polynya efficiency. During this era, sea ice cover and duration have increased in the Ross Sea (RS) region, which is one of the three main sources of AABW, in contrast to opposite trends in the adjacent Amundsen–Bellinghausen Sea2. Simulations performed with state-of-the-art climate models fail to capture such a sea ice increase4. The mechanisms that drive Antarctic sea ice changes, and the respective roles of internal climate variability vs. natural and anthropogenic forcing remain debatable6. Moreover, polar marine ecosystems appear particularly sensitive to changes in the extent and thickness of sea ice7, although much remains to be understood about their past and present responses under rapid environmental changes.Direct knowledge of Antarctic sea ice variability is restricted to the satellite era of the past 39 years (November 1978)8  than pack ice due to the accretion of platelet ice underneath the congelation ice10. Platelet ice formation is strictly related to the efficiency of the coastal polynyas11 to produce sea ice and ice shelf water (ISW), and is mainly driven by katabatic wind persistence. At the seasonal and inter-annual scales, no relationship has been observed between coastal ice and pack ice extensions in the WRS9.Each year, the RS exports by geostrophic winds and ocean currents more than twice its own surface area of pack ice during the average 9-month ice growing period. More than 50% of this pack ice production occurs within the RS and Terra Nova Bay (TNB) latent-heat polynyas8 Fig. , where n13 and is formed by columnar and granular ice plus consolidated platelet ice14. Consolidated platelet ice can form more than 50% of the landfast ice column15 by accreting underneath. The accretion of platelets below congelation ice starts during the mid-austral winter due to the advection of supercooled ISW from underneath floating glaciers and shelf cavities. This process provides an oceanic heat sink for persistent growth of frazil crystals as a first stage for platelet ice formation16. Supercooled ISW has been widely observed along the WRS coast17, associated to thermohaline circulation below ice shelves due to the interaction of high-salinity shelf water (HSSW) melting the ice at the base of the ice shelves. This HSSW mainly forms in the latent-heat polynyas of TNB and RS due to high salt fluxes into the ocean associated with continuous sea ice formation. Only episodically landfast ice can persist for several years along Victoria Land coast (VLC)18. On the other hand, the pack ice is characterised by a thickness of <1 m and is mainly constituted of columnar and granular ice19. This structure is related to the accretion of congelation ice from seawater with ample evidence of a thermodynamic process driven mainly by air and ocean temperatures14.Landfast ice usually forms in early autumn (March–April) and melts back in late summer (January) when the protective surrounding pack ice has waned. A delay of about 45 days is observed between minimum pack ice extent and landfast ice melting/breaking. The annual landfast ice has a thickness of up to 2.5 m2. Reconstruction of past sea ice is based on diatom analyses in marine cores and sea salt sodium (ssNa) in ice cores, while Antarctic climate is inferred from ice core water isotope records. Three gravity cores ANTA99-cJ5, BAY05-20c and BAY05-43c  were collected from the WRS continental shelf. Two cores were collected in the coastal sea ice zone (CSIZ) at Cape Hallett (WRS_CH) and Wood Bay (WRS_WB), in an area characterised by annual landfast ice18, and one was retrieved in the open sea ice zone (OSIZ) at Joides Basin (WRS_JB), where pack ice generally melts and breaks up in early December.This study aims to better understand the main factors controlling WRS sea ice types and dynamic changes during the past millennia on a centennial timescale using three marine core records and two nearby East Antarctic ice core records Fig.  as a ben7. To show this, we compared our new reconstructions to changes in the abundance of three sea ice-dependent fauna  as documented by radiocarbon-dated coastal remains along VLC21.The current study documents contrasting patterns between open and coastal sea ice during the Mid–Late Holocene in the WRS, and an increase in regional polynyas’ efficiency starting from ~3600 years before present . These Holocene changes in pack ice cover, polynya and coastal sea ice are expected to have affected the presence of seals and penguins, animals known to be sensitive to changes in coastal sea ice conditions14C dates performed on acid insoluble organic matter. Overall, the chronology of the three marine cores is robust to within ~600 years, after accounting for uncertainties in the raw dates, regional marine reservoir ages plus local dead carbon (DC) contamination corrections as well as down-core extrapolation. Moreover, the comparisons between ice and marine records have been performed using a 200-year data resampling, that reduced drastically the uncertainty in the correlations. Information on the dating technique, reservoir ages and DC corrections along with the accuracy of the chronologies is detailed in the Methods and Supplementary Note The age models of the marine cores are based on calibrated Fragilariopsis curta group (F. curta and Fragilariopsis cylindrus)24. Both diatoms are sea ice-related species reaching highest abundances both in plankton population and surface sediments in locations that experience 9–11 months of sea ice cover and highly consolidated winter sea ice conditions24. Here we use solely F. curta relative abundances  during events of productivity-triggered nutrient depletion in highly stratified surface waters due to ice melting27. At very high southern latitudes, Thalassiosira antarctica thrives during the summer and forms resting spores (TRS) when sea ice returns in autumn24, therefore tracking long/warm summers followed by cold autumns28. F. curta relative abundances in the two coastal records, WRS_WB and WRS_CH, increase from ~20% before 3.6 ka to ~70–80% over the last 1000 years  ice core records  at moisture source regions31 and can be considered as an integrated tracer of the hydrological cycle31. A southern limit of sea ice would allow a significant contribution from high-latitude cold moisture sources with low d values. Conversely, a northern limit of sea ice would lead to the opposite effect. During the sea ice formation season, a southern shift of sea ice limit and/or an increase in size (efficiency) of latent-heat polynyas would decrease deuterium excess but increase δ18O values due to local (cooler) moisture availability. Conversely, an increase in new sea ice formation is expected to enhance the ssNa content in ice cores.Fingerprints of WRS sea ice and Holocene climate changes can be provided by the nearby Talos Dome (TALDICE) and Taylor Dome (TY) Fig.  ssNa andrds Fig. . The pro32.To identify the ssNa and moisture sources in TALDICE and TY, we analysed the modern air mass trajectories that passed over the sea ice area before reaching those two ice core sites using atmospheric reanalyses and sea ice satellite data Fig. ; Methods35  and new data from TALDICE  to elucidate the link between OSIZ and CSIZ sea ice proxy records from marine and glacial archives. The first component (EOF1) accounts for 43% of the common variance  and the TALDICE ssNa record   and a high positive value for the WRS_JB diatom record  Fig.  and demod and TALDICE ssNa Holocene records  in the OSIZ area of WRS. Conversely, the negative strength of EOF2  and reduced TY ssNa concentrations could be associated with less efficient latent-heat polynyas  and high TY ssNa concentrations were associated with more efficient regional latent-heat polynyas and more pervasive landfast ice. We suggest that increases (decreases) in polynya efficiency, and related ISW formation under floating glaciers/ice shelves, are mostly due to stronger (weaker) katabatic winds and/or seaward position of Ross Ice Shelf (RIS) and Drygalski ice fronts.Both nia Fig. . More prnya Fig. . The proH26 Fig. . As suchF. curta in WRS_JB along with low concentrations of ssNa in TALDICE between 11.8 and 8.2 ka  that may have contributed to RIS recession40. This recession could have formed a calving bay27 over the Joides Basin as suggested by the overwhelming dominance of CRS until 8 ka  records, despite the relative proximity (550 km) of these two coastal core sites. TY δ18O record indicates a cooling at the ice core site throughout the Holocene, while TALDICE δ18O records suggest a warming since 8 ka  result from different source areas and air mass pathways as observed today. Indeed, modern air mass trajectory analysis demonstrate that TALDICE precipitations originate from the Southern West Pacific–East Indian (including Northwestern RS), while TY precipitation is strongly influenced by the RS and TNB polynyas in Southwestern RS  between TALDICE d and F. curta in WRS_JB  and Adélie penguin (Pygoscelis adeliae), as well as the dietary contribution of Antarctic silverfish (Pleuragramma antarcticum) are documented with more than a hundred 14C-dated samples around VLC44  is synchronous to a warm event recorded both in the atmosphere  and in the ocean/sea ice  demonstrating less formation of new sea ice in latent-heat polynyas leading to less platelet ice and thinner coastal sea ice.Our new WRS_JB diatom data suggest that the RIS retreated between 11.8 and 9 ka with a complete opening of WRS by ~7–8 ka, in agreement with previous studiesF. curta relative abundances in CSIZ cores and by high ssNa content in TY during the last millennium, is very likely responsible for the migration of Adélie penguin colonies from the Scott Coast to other sites in southern RS  at 2.8 ka and the disappearance of elephant seal colonies in RS at ~1–0.5 ka20 , corresponding to 1.0–11.8 ka characterised by massive diatomaceous mud with sparse ice-rafted debris clasts. WRS_JB core is generally characterised by low magnetic susceptibility (MS) values . Higher values (from 100 to 400 × 10−6 SI units) correspond to higher sand and gravel content, which are also evident in the X-ray radiographs (not shown). The organic carbon . Relative abundances of reworked diatom species are low with values between 0 and 1.5% of the total diatom assemblage including CRS.WRS_JB core  was retrieved at a water depth of 598 m in the Joides Basin, an elongated trough NE–SW oriented and separated into two parts by a sill located at about 75°S latitudeWRS_CH core  was retrieved at a water depth of 456 m in the outer section of Edisto Inlet, a narrow trough NNE-SSW elongated with a maximum water depth of 500 m, in Cape Hallett area. The core is laminated throughout presenting silt to sandy-silt laminae that are alternated with fluffy diatomaceous mud. Sparse gravel fraction and some carbonate shells are present.−6 SI and exhibiting high variability above 140 cm. Excursions to higher values are due to layers/laminae richer in siliciclastic/volcanoclastic debris. The Corg content  was retrieved at a water depth of 1033 m in the inner part of Wood Bay. Morpho-bathymetric data highlight that sea floor morphology is characterised there by a narrow basin, deeper than 1000 m, oriented WNW-ESE and transversally connected by an 800 m deep sill to the Drygalski Basin. In this paper, we consider the hydrated laminated diatomaceous ooze section (0–409 cm), mainly black in colour alternated with lighter laminae having a cotton-like texture.−6 SI units). The MS values  in correspondence with the increase of terrigenous content. The Corg content .WRS_WB core is characterised by low MS values  from bulk sediment samples, except for one level in WRS_CH core, where a mollusc shell was present  to calculate the modern DC fraction at our core sites. For WRS_JB core lacking a twin box core, a superficial AIOM age of 3781 ± 51 yr, calculated by ref. 48, was used to correct the radiocarbon ages. The core top ages and δ13C value obtained in this study agree with the spatial distribution proposed by refs. 48,49 for the RS. The core top age for WRS_JB is bracketed by surface sediment ages in Joides Basin47 with a standard deviation of ±608 years. The core top age for WRS_WB shows a 200-year younger age than previously reported48 for the WRS. However, the more northern and coastal location of WRS_WB compare to previous studies49 can explain this difference, whereby the local DC contamination is lessened by higher coastal productivity and a South–North decreasing gradient in DC contamination49. The core top age obtained at WRS_CH agrees with those reported by ref. 50 and confirms the decreasing trend of DC contamination towards the northern WRS48. The corrected AMS 14C dates were then converted into calibrated ages using the CALIB REV 7.1.0 calibration program51 and the MARINE13 calibration curve. The conventional, corrected and calibrated AMS 14C data are reported in Supplementary Table The chronology of WRS-CH and WRS-WB marine cores is based on 5 and 6, respectively, accelerator mass spectrometry (AMS) radiocarbon dates measured at the NOSAMS Laboratory of the Woods Hole Oceanographic Institution . For WRS_JB core, four previously published AMS dates have been used14C chronology are reported in the Supplementary Note The arguments that support the −1 for WRS_JB, between 0.64 and 2.87 mma−1 for WRS_CH and between 0.16 and 3.03 mma−1 for WRS_WB , yielding a mean temporal resolution of ~150 years. Core WRS_CH was sampled every 4 cm with a mean temporal resolutions of ~20 years from 1 to 240 cm (0.1–1.4 ka) and ~60 years from 244 to 444 cm (1.5–4.6 ka). The samples were collected at intervals of 4–8 cm in core WRS_WB resulting in mean temporal resolutions of ~30 years from 2 to 301 cm (0–1.2 ka) and ~120 years from 305 to 409 cm (1.4–5.8 ka).52. At least 300 diatom valves in each sample were identified and counted at ×1000 magnification using an Ortholux light microscope with an oil immersion objective lens following the method outlined in ref. 53. The most abundant and ecologically representative diatom species are shown in Fig. Sample preparation for diatom analyses followed the technique described in ref. www.taldice.org). The core provides a palaeoclimate record covering the past 250 ka back to marine isotope stage (MIS) 7.536. Here we consider the uppermost section between 4 and 687 m corresponding roughly to the Holocene period.A 1620 m deep ice core was drilled at Talos Dome in the framework of the Talos Dome Ice Core (TALDICE) project http://isolab.ess.washington.edu/isolab/taylor/) cover the past ~130 ka back to MIS 5e, and is located ~150 km from the western edge of RS, inland of the Transantarctic Mountains. Today, it is characterised by a snow accumulation rate of 65 mm water equivalent per year34.TY ice core records  and water stable isotope records is obtained along 1 m resolution samples (‘bag samples’), which correspond to a mean Holocene temporal sampling resolution of 18 years, ranging between 11 years over the last 2 ka and 24 years over the early Holocene (9–12 ka).58. Na content in Antarctic aerosol is always dominated by sea salt, especially in interglacial periods, showing a minor contribution of dust leachable Na. ssNa has been calculated using non sea salt Ca (nssCa) as crustal marker and a simple two-variable, two-equation system allowing evaluating the ss- and nss-fractions of Na and Ca:Measurements of major ions were performed by means of ion chromatography, with an overall accuracy of the method can be estimated to be around 5%The raw data of the ssNa content obtained from bag samples of the TALDICE ice core for the period corresponding to the Holocene are reported in the Supplementary Fig. 18O) were conducted on continuous bag samples. The TALDICE δ18O  and new δD measurements were conducted using the well-known CO2 (H2)/water equilibration technique. The accuracy of δ18O and δD measurements is ±0.05‰ (1σ) and ±0.7‰ (1σ), respectively, leading to a final precision of ±0.8 ‰ on deuterium excess (d = δD−8 × δ18O). The raw data of the deuterium excess obtained from the bag samples of the TALDICE ice core for the period corresponding to the Holocene are reported in the Supplementary Fig. Measurements of the two water stable isotopes  and they are available every 6 h with 1° latitude–longitude resolution60. We computed one trajectory per day (ending at 12 UTC above the site). Due to the uncertainty associated to trajectories longer than 5 days, which is estimated between 10 and 30% of the travel distance32, the analysis was limited to the first 3 days of the trajectories with the uncertainty reduced significantly. ERA-Interim reanalysis is one of the most reliable atmospheric data sets covering the entire Antarctic Ocean and the last 40 years since the satellite era (1979–present). The most intense katabatic winds, that form the latent-heat polynyas, are influenced by small-scale orography such as coastal valleys. During the last 5 ka, the period analysed in detail in this paper, the geographic configuration of the Western RS and the adjacent ice sheet  were very similar to the modern situation and we could assume similar patterns for latent-heat polynya positions, katabatic wind confluence zones and, consequently, for the main ssNa source areas for each ice core. However, changes in polynya efficiency/katabatic wind feedback mechanisms, sea ice cover/extension and geostrophic wind could have changed the percentage of ssNa from source areas at each ice core site. On the basis of the back trajectory analysis, it was observed that more of the 40% of ssNa at TY and <30% at TALDICE comes from latent-heat polynyas, but it must be considered that any changes in the polynya efficiency during the past could reduce significantly the ssNa coming from the polynyas, in particular for the TY site.A climatology of 5 days backward trajectories ending at 1000 m above ice core site of TALDICE and TY was generated using the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT4) model61. In order to identify air masses presumably responsible for the sea ice-related ssNa transport towards the ice core sites, a selection was carried out taking into account: (i) trajectories which spent at least 10% of their path over the sea and <40% over the continent; (ii) trajectories computed for the period corresponding to the beginning of the sea ice formation, its growth until its maximum extent (March–November); (iii) trajectories which spent at least 3 h consecutively above sea ice, diagnosed for grid cells where SIC fractions are >15%.Each back trajectory was projected on the Sea Ice Concentration field (SIC) and on the 10-m wind field , associating each value along the trajectory path with the nearest SIC and wind speed values−1. The density of events is spatially cumulated for a regular grid of 5° × 1° of longitude and latitude.This selection is refined to calculate the density of back trajectories, which are considered as loaded by ssNa above the sea ice. Loading conditions are defined if the sea ice fraction ranges between 15 and 75 % and the 10-m wind speed >3 m s62.EOF analysis reduces the dimensionality of a data set consisting of a large number of interrelated variables retaining as much as possible of the variation present in a data set. This is achieved by transforming data into a new set of variables, the principal components (EOFs), which are orthogonal to each other, and ordered so that the first few EOFs retain most of the variation present in all the original variablesλ is a diagonal matrix containing the eigenvalues. λj and EOFj represents corresponding eigenvectors, PCj represents principal component time series and highlights the time evolution of the corresponding EOFj. For a detailed description of mathematical fundamental of EOF analysis see ref. 63.As a first step, the original data matrix F is first compressed into a covariance matrix (R), and then the eigenvalue problem is solved as shown below:18O, deuterium excess and ssNa obtained from ice cores and F. curta obtained from marine cores, are used as input data matrix for the EOF analysis, after a 200-year resampling and a standardisation to z-scores. Least squares empirical orthogonal functions approach (LSEOF) is applied64 to decompose the data matrix, which contains missing values due to different lengths of the input records. Several issues were identified with the use of this approach65. A covariance matrix derived from data with different time coverage is not necessarily positive defined and the decomposition via LSEOF can contain negative values of λ leading to a subsequent overestimation of EOFs amplitude and of the amount of explained variance contained therein66. Furthermore, the decomposition of a non-positive defined covariance matrix can lead to a loss of orthogonality between EOFs66. Figure 18O and high positive value for TALDICE and TY deuterium excess proxies, TALDICE δ18O and ssNa, and WRS_CH, WRS_WB, WRS_JB proxies (F. curta). Conversely EOF2 represents a superimposed signal with a high negative score for WRS_CH, WRS_WB proxies (F. curta) and TY ssNa, and high positive WRS_JB proxy (F. curta) and the other records characterised by slight positive values.The time series of δ1 and PC2 time series also reveal different trends (not shown). However, PC time series are less reliable than EOF spatial pattern due to the limited number of overlapping records in the EOFs analysis.PC67 and standardized ((x-mean)/stdev) for the considered period. The linear correlation tables  at the following link: http://isolab.ess.washington.edu/isolab/taylor/data/tdchem.dat; link for water stable isotope data: http://isolab.ess.washington.edu/isolab/taylor/data/tdxs.dat.The data sets for the TY ice core are available at the Taylor Dome Ice Core Project web site, link for chemistry data: Supplementary InformationPeer Review File


Despite important advances in understanding the pathophysiology of AML, therapeutic approaches have not significantly improved patient survival with the exception of ATRA  for acute promyelocytic leukemia (APL),3 CD44 is a transmembrane glycoprotein expressed on both normal and leukemic cells  pathway, frequently dysregulated in AML,8 has not previously been investigated in the context of CD44-signaling in AML. The activation of the Akt signaling pathway results in the loss of control of cell growth and in cancer cell death.9 Because the PI3K/Akt/mTOR pathway is also implicated in sensitivity and resistance to therapy, its blockade is an attractive approach for cancer treatment.Triggering CD44, using monoclonal antibodies (mAbs), is effective at inducing the differentiation and inhibiting the proliferation of many AML subtypes.ic cells , and imp10 in all cell lines tested as early as 5 min that continued to 24 h after treatment, which did not appear to be related to changes in total mTOR expression. Since PI3K and mTOR pathways are suggested to be independently involved in AML cell proliferation, simultaneously blocking both pathways versus only one, should more effectively inhibit the proliferation of leukemic cells.11 We also found that anti-CD44 treatment strongly reduced p-Akt on Thr308, a downstream effector of PI3K in all cell lines   , which i mTORC1) . The mTOf mTORC2 . Altoget13 Phosphorylation of 4E-BP1 is essential for the deactivation of the protein and its subsequent dissociation from eIF4E, which must take place for cap-dependent mRNA translation to proceed. One of the limitations of rapamycin is its failure to inhibit oncogenic protein translation.13 Analysis of the expression of p-4E-BP1 on Ser65 revealed significant baseline levels in AML cells that was inhibited following treatment particularly at 24 h, thereby implying the inhibition of protein translation in these cells  and analyzed for proliferation and for the effect on p-mTOR and p-Akt. Either inhibitor alone, rapamycin or LY294002, induced a significant decrease in p-mTOR , including extracellular signal regulated kinase 1 and 2 (ERK1/2) and src family kinases (SFKs), including Lyn, Fgr and Hck.21 An attractive candidate for the cross-talk between CD44 and mTOR is the non-receptor tyrosine kinase Syk, which has emerged as a critical regulator of mTOR in AML blasts.22The mechanisms through which CD44 ligation inhibits mTOR are yet to be elucidated. Although LY290004 inhibited Akt phosphorylation on Thr308 as well as the cell proliferation, it did not inhibit mTOR phosphorylation as significantly as A3D8. This suggests that if PI3K inhibition is involved in the mTOR inhibition induced by the anti-CD44-mAb, it is not the only pathway responsible for the inhibition of mTOR phosphorylation 3 (KG1a is a leukemic cell line whose inhibition of proliferation but not differentiation (contrary to HL60 and THP-1 cells) is induced by anti-CD44-mAb treatment3 . Interes3  legend. 23 along with inhibiting protein translation, reinforces the idea of using anti-CD44-mAbs in therapeutic strategies for leukemia.In summary we provide compelling evidence that the inhibition of proliferation, and in some cases the induction of differentiation, of AML cells induced by anti-CD44-mAb treatment is accompanied by a marked decrease in the phosphorylation of the mTORC1 and mTORC2 complexes, which is strongly correlated with the inhibition of the PI3K/Akt pathway. Since perturbations in the PI3K and mTOR pathways are commonly observed in leukemia, blocking two major players of the PI3K/Akt/mTOR pathway


Otocyon megalotis, Canidae, Carnivora, Mammalia) is one example; however, its developmental origin is not clear. In some canids (Canidae), individual variation exist as supernumerary molar M4. This study focuses on the acquisition of the lower fourth molar in canids and proposes that the inhibitory cascade model can explain its origin.An increase in tooth number is an exception during mammalian evolution. The acquisition of the lower fourth molar in the bat-eared fox  affected by inhibition/activation dynamics during development, were compared between individuals with and without supernumerary molar (M4).Occlusal view projected area of lower molars was determined from 740 mandibles obtained from 2/M1 score in Canis latrans, M3/M1 score in Nyctereutes procyonoides, and M2/M1 and M3/M1 scores in Urocyon cinereoargenteus compared to individuals of these species that lacked supernumerary molars.Possession of a supernumerary molar was associated with significantly larger MWe propose that, in canids, the supernumerary fourth molar is attributable to reduced inhibition and greater activation during molar development. In the bat-eared fox, altered inhibition and activation dynamics of dental development during omnivorous-insectivorous adaptation may be a contributing factor in the origin of the lower fourth molar. The dental formula in the bat-eared fox is I 3/3, C1/1, P 4/4 M 3–4/4–5, with these animal usually possessing an upper M3 and lower M4 that ancestral Canidae lacked  . Some spof teeth . Howevere lacked , and thie lacked .Bat-eared foxes are primarily insectivorous , with a 4 in the bat-eared fox was investigated based on examination of supernumerary teeth in related species.Individual variations in tooth number (supernumerary and missing teeth) have been reported in many mammals e.g. . In some3 and M4 along the normally aligned tooth row in coyote (Canis latrans) (Urocyon cinereoargenteus)   and graygenteus) . Wolsan essed M3 , the lasr molars .2 and M3), which makes them larger in relation to M1, whereas inhibition molecules secreted from M1 suppress distal molars growth (M2 and M3)  result in a pattern of M1 > M2 > M3, M1 = M2 = M3, or M1 < M2 < M3  to explain relative molar size among three lower molars in mammals as resulting from the balance of inhibition and activation molecules during dental development . In this and M3) . Accordi M2 < M3 . This mo M2 < M3 .3 during murine evolution, such that greater inhibition and lower activation in experimental mice organs resulted in the disappearance of M3, coinciding with changes in the M2/M1 and M3/M1 ratios. This corresponds to the dentition of the murine species Hydromys chrysogaster, which lacks the M3 (3 loss) and the IC model in canids, in which M2/M1 scores of individuals that lost M3 were lower (indicating higher inhibition and lower activation) than the scores in normal individuals in local populations of raccoon dog (Nyctereutes procyonoides) and arctic fox (Vulpes lagopus)  appeared in mice following suppression of inhibition molecules, indicative of an increase in molar number and altered inhibition/activation dynamics.The IC model can also explain the loss of Ms the M3 . Asaharalagopus) . Evoluti4).The working hypothesis for this study is that the fourth lower molars in the bat-eared fox and the supernumerary molar in some other canid species are generated by reduced inhibition and greater activation during dental development. This was tested by comparing the relative size of molars in locations of three canid species with some individuals possessing a supernumerary molar , 153 Nyctereutes procyonoides , and 136 Urocyon cinereoargenteus  were examined. The collection of C. latrans was chosen based on an earlier study using the U.S. collection, which reported the presence of a supernumerary molar M4 in some specimens . The projected area is defined as molar size, as described by  and that of M3 to M1 size (M3/M1) were calculated to compare relative sizes among molars. According to the IC model , with analyses performed using Minitab 14 statistical software . For data of the bat-eared fox Otocyon megalotis in Mandible specimens (dentary bones and molars) of 451 pecimens . Presencribed by . For speIC model , lower iC. latrans , four individuals of N. procyonoides , and five individuals of U. cinereoargenteus  possessed an M4 in the normally aligned molar row , with the exception of M2/M1 scores in the Gifu samples of N. procyonoides  did tend to possess a supernumerary molar M4  and the IC model reported by 1 size found in C. latrans  have reported no M4 molar in Japanese samples  possess low M2/M1 scores and the more omnivorous species (such as N. procyonoides or U. cinereoargenteus) have higher scores  than the carnivorous or omnivorous species, with higher M2/M1 and M3/M1 scores reflecting low inhibition and high activation during molar development. The present study is consistent with the hypothesis that low inhibition and high activation is a cause of M4 generation. Therefore, M4 is considered to be an adaptive trait providing a larger total occlusal surface area to enable the canid to digest a large amount of insects, as discussed by 1, M2, and M3) than its ancestor, providing an evolutionary force supporting this adaptation to the insectivorous diet. Then, the inhibition/activation pattern generates M4 by chance. M4 is also an adaptive trait for insectivorous diet providing larger occlusal surface area and greater grinding function, therefore, presence of M4 is naturally selected and fixed in the ancestral lineage of the bat-eared fox. While no fossil canid species has been to found to possess an M4, several cases of a small supernumerary M3 has been reported in an individual of the Prototocyon genus (Otocyon (r scores . In addielopment . Accordion genus , which m(Otocyon . This su4 observed in several canid species is influenced by low inhibition and high activation during molar development, with the presence of M4 in the bat-eared fox O. megalotis originating from low inhibition and high activation due to evolutionary pressure relating to an insectivorous diet.The presence of a supernumerary molar M10.7717/peerj.2689/supp-1Supplemental Information 1Click here for additional data file.