In recent years, site-specific antibody drug conjugates (ADC)s have been in great demand because they have an expanded therapeutic index compared with conventional ADCs. AJICAP™ technology is a chemical conjugation platform to obtain site-specific ADCs through the use of a class of Fc-affinity compounds. Promising results from early technology development studies led to further investigation of AJICAP™ ADC materials to obtain site-specific and homogeneous drug antibody ratio (DAR) ADCs. Here we report site-specific conjugation followed by a preparative hydrophobic interaction chromatography (HIC) purification strategy to obtain purified “DAR = 1.0” and “DAR = 2.0” AJICAP™ ADC materials. Optimization of the mobile phase conditions and resin achieved a high recovery rate. In vitro biological assay demonstrated the target selective activity for purified homogeneous DAR ADCs. These results indicate the ability of a HIC purification strategy to provide “DAR = 1.0” and “DAR = 2.0” AJICAP™ ADCs with considerable potency and target selectivity. Recent years have seen rapid growth in the research and development of antibody drug conjugates (ADCs), with 7 ADCs approved for clinical use by the Food and Drug Administration (FDA) and over 85 ADCs currently in clinical development ,2,3. AllSeveral advancements have been made regarding site-specific conjugation technologies in efforts to overcome the issues derived from the heterogeneity of traditional ADCs . Our resIn contrast to conjugation methodologies, literature reports examining purification techniques to separate DAR species have been extremely limited to date . In 2004This report described the purification of an ADC with a particular DAR, but not a homogeneous site-specific ADC as the group used traditional cysteine-based conjugation, which led to a distribution of drug-linker attachment sites. For example, theoretically there are three species of ADC with DAR = 2 which could be produced by this approach, as shown in The stochastic distribution of traditional ADCs is known to have a negative effect on the therapeutic index. In 2013, Rajpal, Strop, and coworkers reported that the conjugation site has a considerable impact on the stability and properties of ADCs . These rIn addition to creating ADC with DAR = 2, we are interested in developing methodologies to produce homogeneous ADCs with DAR = 1, which have the potential to expand the therapeutic index of hydrophobic or highly potent payload-based ADCs. In recent years, highly potent payloads such as pyrrolobenzodiazepine (PBD) dimers have received a large amount of attention . The modThis approach demonstrated a robust methodology combining site-specific conjugation and preparative HIC purification to obtain “ideal” homogeneous site-specific ADCs.®) was purchased from Roche Pharmaceutical Company (Switzerland). Maleimide-C6-valine-citrulline-monomethyl auristatin E  was purchased from Abzena . The peptide reagent (1) was provided by Ajinomoto Co., Inc. All other chemicals were purchased from Sigma-Aldrich . All cell lines (HCC-1954 and NCI-N87 cancer cell lines) were purchased from ATCC .Human IgG1, trastuzumab (Herceptin2). Treatment of 2 by Tris (2-carboxyethyl)phosphine (TCEP) clipped off peptide moiety from 2 involving cleavage of interchain disulfide bonds. The resulting linker cleavage product was converted to product (3) by dehydroascorbic acid (DHAA) oxidation. Conjugation of 3 to MC-VC-MMAE was performed to produce trastuzumab-AJICAP™-MMAE (4A). Next, the buffer exchange of this solution was conducted by an Amicon Ultra centrifuge filter (10 kDa MWCO) to afford trastuzumab-AJICAP™-MMAE (4A) in formulation buffer .Unpurified trastuzumab-AJICAP™-MMAE (4) was synthesized according to our previous procedure . PeptideUnpurified trastuzumab-AJICAP™-ADC (4A) was purified by HIC column , prepacked by ToyoPearl Phenyl-650S HIC resin (Tosoh Bioscience) and attached to an AKTA Pure system . All proThe concentration of ADCs was determined by the slope spectroscopy method with a SoloVPE system as previously reported . Recover4)2SO4, 25 mM NaHPO4/NaH2PO4 (pH 6.0); mobile phase B (MPB) = 25 mM NaHPO4/NaH2PO4 (pH 6.0) with 25% IPA v/v). The absorbance was monitored at 280 nm (reference wavelength at 450 nm). Each ADC  was injected into the system and eluted over a 27 min run consisting of a 2 min isocratic hold at 0% MPB, a 20 min linear gradient from 0% to 100% MPB, a 2 min wash using 100% MPB, and a 3 min re-equilibration at 0% MPB.HIC-HPLC analysis was performed based on previously reported . Each ADRP-HPLC analysis was performed based on previously reported . Each sam/z range of 1000–4000. For reduction deconvolution, we used a mass range of 20,000–60,000 and a limited m/z range of 1000–3000. Moreover, we used DAR Calculator software (Agilent) to determine the PAR and DAR.Quadrupole time-of-flight mass spectrometry (QTOF MS) analysis was performed on a PLRP-S 2.1 × 50 mm, 1000 Å, 5 μm column (Agilent), connected to an Agilent 1260 HPLC system with an Agilent 6550 QTOF system containing a binary gradient pump, a temperature-controlled column compartment, and an autosampler. The elution conditions were as follows: mobile phase A = 0.1% formic acid, 0.01%, trifluoroacetic acid (TFA), 2% acetonitrile in water; mobile phase B = 0.1% formic acid and 0.01% TFA in acetonitrile; gradient 0–2 min isocratic hold at 20% MPB; 2–12 min, 20–50% B; 12–15 min, 95% B, 15–15 min, 20% B; flow rate = 0.3 mL/min. The absorbance was measured at 280 nm. Automatic data processing was performed with MassHunter BioConfirm software (Agilent) to analyze the intact and reduced MS spectra. For intact deconvolution, we used a mass range of 100,000–180,000 and a limited 4/NaH2PO4, 250 mM NaCl, 10% v/v IPA, pH 6.8 . The absorbance was monitored at 280 nm (reference wavelength at 450 nm). Each ADC  was injected into the system and eluted over an 11 min run consisting of MP.Size exclusion chromatography (SEC)-HPLC analysis was performed based on previously reported . Each ADTrastuzumab-AJICAP™-ADCs  and trastuzumab were analyzed using PC3, HCC-1954, and NCI-N87 cancer cell lines as previously reported .Site-specific trastuzumab-AJICAP™-MMAE was synthesized by the first generation AJICAP™ procedure (a well-established approach that in this case generated a defect-DAR ADC mixture), which provided gram-scale ADC materials shown in Next, preparative HIC purification of the resulting AJICAP™-ADC was conducted with an AKTA Pure chromatography system. For the purification investigation, prepacked columns of various resins  and HER2 antigen-overexpressing cell lines (NCI-N87: gastric cancer cell and HCC1954: breast cancer cell) were evaluated based on our previous study as shown Displayed in To reduce the heterogeneity of ADCs, a combination strategy of chemical site-specific conjugation and preparative HIC purification was developed. Very recently, Müller and coworkers reported resin screening for the purification of an ADC-mimic . This reChemical conjugation approaches for the site-specific construction of ADCs are of great interest to the oncology therapy community. These methods benefit from straightforward chemistry, manufacturing, and control of the resulting ADCs. Preparative protein chromatography is commonly applied to the purification of biological drugs at a manufacturing-scale. Therefore, the combination of chemical site-specific conjugation and preparative HIC purification can be considered to be a robust strategy to produce next generation ADCs. The feasibility study described herein can serve as a useful guide in the preclinical process development of site-specific and homogenous DAR ADCs. The reported cytotoxic evaluation using three different cell lines in the present manuscript showed that the compounds had considerable potency and target selectivity after purification. The biological and analytical evaluations described herein are the first report regarding the potency of a homogeneous DAR = 1.0 ADC produced by a chemical site-specific approach. We are confident that continued conjugation innovations and strategies in protein purification will aid in the further development of site-specific ADC technologies. Homogeneous DAR = 1.0 ADCs have the potential to be applied to highly potent ADC payloads to overcome the situations of a narrow therapeutic window, and these initial studies provide support for our further biological evaluations to obtain more efficacious ADCs.


Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off‐target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly “rigid protein” that substantial active‐site dynamics have generally remained unrecognized, despite thousands of medicinal‐chemistry studies on this model over decades. Comparing cryogenic X‐ray structures, solid‐state NMR on micro‐crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational‐exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines. Human carbonic anhydrase II (hCAII) plays an important role as a rigid model protein in medicinal chemistry. Under physiological conditions in solution, NMR spectroscopy combined with molecular dynamics simulations demonstrates that the active site shows pronounced open/close conformational exchange, undetected in crystallography‐based work. Innumerable structural studies, mainly via X‐ray,Drug discovery, the basis for successful disease treatment in our society and a multi‐trillion dollar business, relies on screening campaigns in combination with medicinal chemistry to improve binders with respect to their pharmacological properties. Besides the affinity and specificity to the target, tweaking lead compounds with respect to binding kinetics is important to endow them with appropriate efficacies and tolerable toxicity properties under physiological conditions.Aided by solid‐state NMR studies providing complete active‐site assignments,Next, we determined the protein structure under solution conditions Figure , with anR2 rates still elevated up to 37 s−1  with unclear origin widely spread over the pocket for crystalline hCAII at room temperature.12c Where Figure –D. The c−1 on the activation energy for catalysis and a remaining 105 turnovers per second.Next, in order to mechanistically assess the nature of the active‐site conformational exchange in more detail, we carried out molecular dynamics (MD) simulations with GromacsR1 and hetNOE data. In the uninhibited case, fast‐timescale motion is present only at the tips of some external loops, which are associated with structural deviations to the crystalline state (see above). hetNOE values in the absence of inhibitor scatter around 0.8 for the whole sequence, again showing slightly elevated fast‐timescale motion for the loop around T198.  By contrast, upon inhibition, fast‐timescale motion  is observed for residues in large parts of the primary sequence. Statistics and differences, liganded vs. unliganded form, are shown in Figure 2 binding to the active site as the natural substrate. Its affinity is approximately 100x lower than the inhibitor, such that these data only show subtle effects. The trends, however, seem to be in line with the sulfonamide as a high‐affinity  substrate analogue.Drug discovery on CAs has afforded manifold sulfonamide inhibitors, which are known to replace the Zn‐bound catalytic water molecule. Hence, they also impair the conserved water network, one of the features of the CA core.1, 16 − in the inhibitor‐free form  is easily opened at room temperature in the absence of a crystal lattice, such that the active‐site loop undergoes pronounced open/close dynamics. The timescale of the active‐site loop motion is further modulated by the hydrophobic interactions with the N‐terminus, which couples the dynamics of these two structural regions. As such, the protein has the possibility to dynamically adjust the active site towards a suitable geometry. The conformational‐exchange timescales are comparable to the catalytic turnover rate of the enzyme, underlining its likely relevance for biological activity. The sampling of open and closed conformations in the absence of a substrate may be advantageous for substrate intake and also adds to understanding hCAII substrate flexibility.Our various observations unambiguously demonstrate the existence of conformational‐exchange backbone dynamics in the active site of hCAII under close‐to physiological conditions. This contradicts the conclusions from X‐ray crystallography‐based studies17, 18Here, we have demonstrated important differences for properties in the active site of hCAII, unmatched “rigid” model system for drug discovery, under biologically representative conditions in solution compared to previous crystallography‐based studies. Using NMR relaxation, relaxation dispersion, RDCs, and MD simulations, we have demonstrated that μs timescale backbone conformational exchange between open and closed forms exists for the important active‐site loop under native conditions. The active‐site plasticity hitherto undetected for this prominent target calls for awareness upon assessing enthalpy and entropy of binding and in structure‐guided lead optimization via standard approaches.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The mean correction rate immediately after surgery was 60.8%, and the correction loss rate at the observation end point was 4.8%. The Cobb angle of the lumbar curve was significantly improved immediately after surgery, and the correction persisted until 10 years post-operatively. Remarkable gains were observed for most Scoliosis Research Society-22 patient questionnaire sub-scores at the final follow-up versus preoperative assessments. In conclusion, good correction of the AIS deformity by skip pedicle screw fixation was well maintained over a long follow-up period of 10 years, with clinically meaningful gains in Society-22 patient questionnaire sub-scores.Skip pedicle screw fixation for adolescent idiopathic scoliosis (AIS) requires fewer screws and can reduce the risk of neurovascular injury as compared with segmental pedicle screw fixation. However, the long-term impact of screw number reduction on correction and clinical results is unclear. This study examined the 10-year post-operative outcomes of skip pedicle screw fixation for patients with AIS. We reviewed the outcomes of 30 patients who underwent skip pedicle screw fixation for AIS. Radiological and clinical findings were assessed before and immediately, 2 years, and 10 years after surgery in the remaining 25 patients. The mean Cobb angle of the main curve preoperatively and immediately, 2 years, and 10 years post-operatively was 59.4°, 23.4°, 25.8°, and 25.60°, respectively, and was significantly improved at all post-surgical time points (all  Segmental pedicle screw fixation using numerous thoracic pedicle screws as described by Suk et al.  is a useThe purpose of this study is to examine if the corrective position retention and good clinical outcomes of skip pedicle screw fixation are maintained even at 10 years after surgery. To further validate the usefulness of skip pedicle screw fixation for AIS, we examined the radiological and clinical findings of patients after a long observation period of at least 10 years.doi.org/10.5281/zenodo.4295859).This study was conducted at our institution. We earlier described the 2-year results of skip pedicle screw fixation in 62 consecutive cases of AIS . Among 3Pedicle screw insertion into the upper and lower ends of the instrumentation area was performed bilaterally. Screw placement in the vertebral levels other than the upper instrumented vertebra, lowest instrumented vertebra, and apex was judged based on the size and rigidity of the curve and was skipped when possible. Screw insertion was skipped near the apical vertebrae of each curve and in the junctional zone between structural and compensatory curves that were not close to fused end vertebrae. We excluded pedicles with an outer diameter of less than the thinnest screw diameter during navigation planning. Pedicle screws were routinely inserted near the concave apical vertebrae. For cases in which a 4.5 mm-diameter pedicle screw appeared difficult to insert in navigation planning, tape was used instead. However, in cases where only one vertebra was skipped, no tape was used. Since pedicle diameter is wider near the convex vertebrae, we basically used pedicle screws and skipped one vertebra per inserted vertebra. Screw holes were made using a Kotani probe  with CT-p-value of <0.05 was considered statistically significant.The first author (M.U.), who was not associated with any surgery in the cohort, performed the evaluation. Cobb angle  and thorThe cohort’s preoperative radiological and clinical features are summarized in p < 0.001)  , Table 2p < 0.05)  , Table 2The Cobb angle of the lumbar curve and LIV tilt were significantly improved immediately after surgery, which persisted until 10 years post-operatively , Table 2p < 0.05)  . At the  < 0.05) .p = 0.004). The JOABPEQ scores for low back pain in patients with and without lumbar decompensation were comparable, with values of 91.4 ± 19.2 and 85.7 ± 21.8, respectively (p = 0.58). The JOABPEQ score for low back pain in patients with adding-on was significantly higher than in patients without it . The respective JOABPEQ scores for low back pain in patients with and without early degeneration were similar, corresponding to 62.0 ± 32.9 and 90.2 ± 17.4 (p = 0.27). The mean VAS score for low back pain was 19.6 ± 5.4 (range: 0−80) and was zero in 13 (52%) patients. Bone union was confirmed by CT in all cases. Screw breakage without symptoms requiring surgical revision was seen in two patients. No severe neurovascular complications from surgical invasion or indication for surgical revision were recorded.JOABPEQ scores at 10 years after surgery were 86.8 ± 4.2 for pain-related disorder, 94.0 ± 2.4 for lumbar spine dysfunction, 95.8 ± 2.7 for gait disturbance, 84.4 ± 3.6 for social life disturbance, and 72.9 ± 3.4 for psychological disorder . Three .Ishikawa et al., reported that the final Cobb angle of the thoracolumbar/lumbar curve was significantly correlated with the immediately post-operative LIV tilt . On the The JOABPEQ domain scores in our cohort were all considerably higher than the reference values of low-back-pain patients  Table 4Table 4. Regarding long-term radiological results, the main thoracic curve Cobb angle improved from 51° to 16°, with a correction loss rate of 5% in a five-year study on segmental pedicle screw fixation for thoracic scoliosis . HoweverWith regard to the long-term clinical results of surgical AIS treatment, there are several reports on the Harrington method, posterior fixation with the rod and hook system, and anterior fusion, and few on pedicle screw fixation. In a comparative control study using the SRS-24, AIS patients undergoing correction surgery by the Harrington method had findings equivalent to those of healthy subjects for all parameters, and the magnitude of the scoliotic angle was not associated with quality of life (QOL) scores . In a siWe lastly examined whether the SRS-22r score changes in our cohort were clinically meaningful. In earlier studies on MCID for clinical domain outcomes, MCID was 0.20 for pain (area under the receiver operating characteristic curve (AUC) = 0.723), 0.08 for function (AUC = 0.648), and 0.98 for self-image (AUC = 0.629) . The chaNewton et al., noted that posterior segmental instrumentation for AIS could significantly decrease thoracic kyphosis . Such a The main limitations of this study were no control group, a limited sample size, and a retrospective design. No blinding of the assessor was also a limitation of this study. Although five patients were lost to follow-up, the resulting 83.3% follow-up rate was considered satisfactory based on Solberg et al., reporting that a follow-up loss of 22% did not bias conclusions about the effects of treatment . Thus, oSkip pedicle screw fixation for AIS provided significant and sustained radiological and clinical improvements at 10 years after surgery without serious complications.


Several anesthetic agents are used in cesarean sections for both regional and general anesthesia purposes. However, there are no data comparing the in vivo effects of propofol, sevoflurane, and dexmedetomidine on the contraction of the myometrium in pregnant rats. The aim of this study was to investigate the effect of these anesthetic agents on myometrial contraction and elucidate the underlying mechanisms.Contraction force and frequency changes in response to propofol, dexmedetomidine, or sevoflurane were evaluated in vivo and in vitro. To test the effect of arachidonic acid on myometrial contraction enhanced by dexmedetomidine, changes in myometrial contraction with dexmedetomidine after administration of indomethacin were evaluated. The amount of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in the membrane fraction was expressed as a percentage of the total fraction by Western blot analysis.This study demonstrated that dexmedetomidine enhances oxytocin-induced contraction in the myometrium of pregnant rats, whereas propofol and sevoflurane attenuate these contractions. The dexmedetomidine-induced enhancement of myometrial contraction force was abolished by the administration of indomethacin. Propofol did not affect oxytocin-induced MYPT1 phosphorylation, whereas sevoflurane attenuated oxytocin-induced MYPT1 phosphorylation.Inhibition of myofilament calcium sensitivity may underlie the inhibition of myometrial contraction induced by sevoflurane. Arachidonic acid may play an important role in the enhancement of myometrial contraction induced by dexmedetomidine by increasing myofilament calcium sensitivity. Dexmedetomidine may be used as a sedative agent to promote uterine muscle contraction and suppress bleeding after fetal delivery. Several anesthetic agents are used in cesarean sections for both regional and general anesthesia purposes. Some anesthetic agents have an inhibitory effect on myometrial contraction and may cause atonic bleeding . A close. reported that the inhibition of contraction by propofol involves inhibition of increases in the intracellular concentration of free Ca2+. In other smooth muscles, for example, vascular smooth muscle tone is regulated by changes in both the intracellular Ca2+ concentration and myofilament Ca2+ sensitivity  ethane sulfonic acid) (50 mM), adjusted to a pH of 7.5, Triton X-100 (1%), NaCl (50 mM), NaF (50 mM), EDTA (5 mM), sodium pyrophosphate (10 mM), phenylmethanesulfonyl fluoride (1 mM), Na3VO4 (1 mM), leupeptin (100 µg/mL), and aprotinin (10 µg/mL). Homogenates were centrifuged at 1.0 × 104 g for 15 min at 4 °C, and the supernatant was collected. The protein concentration of each supernatant was determined using a Pierce BCA Protein Assay kit . Equal amounts of total protein were used for every sample in each experiment. Strips were treated with anti–β-actin antibody (1:1000) as a loading control. Proteins were separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The membranes were treated with anti-MYPT1 antibody (1:1000) and anti-phosphorylated MYPT1 antibody (1:1000). The densities of the immunoreactive bands were determined using enhanced chemiluminescence , and the data were analyzed using an image analysis system . The amount of phosphorylated MYPT1 in the membrane fraction is expressed as a percentage of the total fraction.The relationship between the effects of sevoflurane or propofol on uterine muscle contraction and MYPT1 activity was investigated using Western blot analysis. Pregnant Wistar rats were anesthetized with sevoflurane and sacrificed by decapitation. Myometrial tissue strips were collected and treated with oxytocin in the presence or absence of propofol . For statistical evaluations, data were analyzed by paired All uterine strips demonstrated spontaneous contraction in the organ baths. Figure There was no difference in the frequency of myometrial contraction induced by any anesthetic agent between the two concentrations examined. The higher concentration of dexmedetomidine significantly enhanced the myometrial contraction force Fig. . The admP < 0.05). Propofol did not affect oxytocin-induced MYPT1 phosphorylation , whereas sevoflurane attenuated oxytocin-induced MYPT1 phosphorylation   was used in some previous studies , the dos2+ sensitivity may have inhibited the myometrial contraction induced by sevoflurane. Arachidonic acid may play an important role in the enhancement of myometrial contraction induced by dexmedetomidine via enhancement of myofilament Ca2+ sensitivity. Dexmedetomidine could thus be used as a sedative agent after fetal delivery to promote uterine muscle contraction and suppress bleeding.In summary, this study demonstrated that dexmedetomidine enhances oxytocin-induced contraction in the myometrium of pregnant rats, whereas propofol and sevoflurane attenuate contraction. The mechanism underlying the suppression of myometrial contraction differed between propofol and sevoflurane. Inhibition of myofilament Ca


A number of feedbacks regulate the response of Arctic sea ice to local atmospheric warming. Using a realistic coupled ocean‐sea ice model and its adjoint, we isolate a mechanism by which significant ice growth at the end of the melt season may occur as a lagged response to Arctic atmospheric warming. A series of perturbation simulations informed by adjoint model‐derived sensitivity patterns reveal the enhanced ice growth to be accompanied by a reduction of snow thickness on the ice pack. Detailed analysis of ocean‐ice‐snow heat budgets confirms the essential role of the reduced snow thickness for persistence and delayed overshoot of ice growth. The underlying mechanism is a snow‐melt‐conductivity feedback, wherein atmosphere‐driven snow melt leads to a larger conductive ocean heat loss through the overlying ice layer. Our results highlight the need for accurate observations of snow thickness to constrain climate models and to initialize sea ice forecasts. Arctic sea ice sensitivity to atmospheric warming is computed using a numerical adjoint, revealing warming can lead to lagged sea ice growthSnow melt—in response to early warming—is the key preconditioner, enabling large conductive ocean heat loss to support lagged ice growth Our quantity of interest is the monthly mean sea ice volume  in the evolution of the heat budget for the ocean‐ice‐snow system. Time series of heat content change, integrated over the full depth of the relevant  component is shown in Figure The pattern of the imposed warm perturbation, Figures The net vertical heat flux , and  ocean‐atAn enhanced conductive heat flux  in snow and ice resistance at the start Figures 1 and 4b2To ascertain the key role of snow in sustained ice growth in response to atmospheric warming, we repeated the adjoint sensitivity calculations in another simulation where snow was removed from the initial conditions and precipitation was turned off at runtime to prevent new snow from forming. In this snow‐free Arctic experiment, the positive sensitivity of ice volume with respect to air temperature seen in WARM is absent (figure not shown). Atmospheric warming leads to enhanced sea ice melt at short lag. The sea ice slowly recovers through the following growth season, never overshooting its original (unperturbed) volume.4Using a coupled ocean‐sea ice GCM and its adjoint, we have investigated the space and time‐evolving sensitivity of total sea ice volume in the Arctic to changes in near‐surface atmospheric temperature. Counter‐intuitively, we find that near‐surface atmospheric warming can lead to lagged sea ice growth. Specifically, we identify extended regions where warming at the end of the melt season enhances subsequent ice growth.The responsible mechanism is a negative conductivity feedback, wherein enhanced summer melt preconditions the system for faster growth in the following winter via enhanced conductive heat loss. Previous studies have focused on the importance of early ice thinning in this feedback (i.e., the negative sea ice‐conductivity feedback, Bitz & Roe, Unambiguous detection of negative snow‐melt‐conductivity feedback is enabled through calculation of time and space‐varying ice volume sensitivities to surface air temperature. To our knowledge, this is the first published adjoint‐based investigation suggesting that warming can lead to sea ice growth. We note this feedback is not evident in the adjoint‐based assessment of Arctic ice volume change by Koldunov et al. , who appWe have focused on the response of Arctic sea ice volume to atmospheric warming. We did not address the response of Arctic ice volume to near‐surface ocean warming, although this has received significant attention in the literature in recent years, amid growing evidence of enhanced upper ocean radiative heating of the Arctic Ocean (Moore et al., To elucidate the importance of the insulating snow layer, we imposed an artificial surface warming perturbation Figure , purposeSnow thickness at the beginning of the sea ice growth season determines the strength of the subsequent conductivity feedback described in this study. In turn, snow thickness depends on a variety of factors (see Figure 1 Webster et al., Supporting Information S1Click here for additional data file.Data Set S1Click here for additional data file.Data Set S2Click here for additional data file.Data Set S3Click here for additional data file.Data Set S4Click here for additional data file.


We find that in 67%, 88% and 52% of the obliquity cycles during the early, middle and late Quaternary, respectively, a glacial termination is realized leading to irregular appearances of new interglacials during various parts of the last 2.6 Myr. This finding suggests that the proposed idea of terminations leading to new interglacials in the Quaternary as obliquity driven with growing influence of land ice volume on the timing of deglaciations during the last 1 Myr might be too simple. Alternatively, the land ice-based definition of interglacials needs revision if applied to the entire Quaternary.Glacial/interglacial dynamics during the Quaternary were suggested to be mainly driven by obliquity (41-kyr periodicity), including irregularities during the last 1 Myr that resulted in on average 100-kyr cycles. Here, we investigate this so-called Mid-Pleistocene Transition via model-based deconvolution of benthic  This study presents a new definition of interglacials during the Quaternary. The authors find the appearance of interglacials is in general following the 41-kyr cycle of obliquity with various exceptions, suggesting a more complex physical mechanism triggering glacial terminations. However, orbital theory alone cannot explain the reconstructed evolution from a 41-kyr dominated glacial cyclicity earlier in the Quaternary towards a 100-kyr periodicity during the last 1 Myr5. For an explanation of this so-called Mid-Pleistocene Transition (MPT), feedbacks in the climate system have to be considered. Prominent candidates of those feedbacks are land ice dynamics7, possible driven by regolith removal9 and the gradual decline in atmospheric CO2 concentrations during glacial minima12. Hypotheses to explain this MPT have been the subject of many studies throughout the last four decades26. One of the most recent approaches to explain the observed pattern during the MPT24 (T17 in the following) developed a simple rule by which the onset of new interglacial periods during the last 2.6 Myr are identified based on caloric summer half-year insolation at 65°N, whereby it assumes that deglaciations (melting of land ice) mainly depend on local summer insolation. In this concept, the amount of energy necessary to trigger a deglaciation which leads into an interglacial decreases with the amount of time since the previous deglaciation as it is assumed in T17 that ice-sheet instability increases with time.Changes in Earth’s orbit are understood to be the fundamental reasons for the ice age dynamics during the Quaternaryδ18O stack4, including some tests of these methods with alternative benthic δ18O records. This definition of interglacials agrees for the last 800 kyr with findings of a more rigorous community effort27 based on the analysis of various climate variables. As consequence of this new definition of interglacials24, no regular 100-kyr periodicity of interglacials occurs after the MPT. Instead, during the last  ~1 Myr, the regular appearance of interglacials every 41-kyr following the pacing of obliquity before the MPT is replaced by an irregular appearance of interglacials with  ~80-kyr or  ~120-kyr cyclicity of interglacials . The latitudinal distribution of land ice across the northern hemisphere would need to include insolation at all latitudes and, if investigated within an energy balance scheme30, across the full year. Our findings will be discussed in the light of uncertainties and available independent evidences, including the role of atmospheric CO2 concentration. We find that the glacial/interglacial amplitudes of northern hemisphere land ice variability outside of Greenland are much smaller prior to than after the MPT. At the same time the contributions from deep ocean temperature and ice volume in Antarctica and Greenland to this change in glacial/interglacial amplitude across the MPT in the corresponding benthic δ18O stack are much smaller. According to our new definition of interglacials their appearance is in general following the 41-kyr cycle of obliquity with various exceptions throughout the last 2.6 Myr, suggesting a more complex physical mechanism triggering the glacial terminations.In the following, we will analyse land ice dynamics obtained in the framework of the inverse deconvolution of the LR04 27) by passing thresholds in the benthic LR04 δ18O stack, which has been detrended before 1.5 Myr BP. Because benthic δ18O is globally integrating all land ice changes, also on Greenland and in Antarctica, and because it additionally contains a contribution from deep ocean temperature change, the record has been detrended in T17 in order to approximately correct for these secondary effects. Here, we are in the position to define interglacials as originally meant in T17 and as proposed as an objective criterion for the last 800 kyr27: by changes in land ice volume in North America and Eurasia  is a continued interglacial. The only two exceptions are periods containing MIS 27 (~1 Myr BP) and MIS 33 (~1.11 Myr BP). According to our definition, the obliquity cycle around 1 Myr ago should be classified as interglacial, but it fails  to be so in T17. Around 1.11 Myr BP, including MIS 33, the ice volume very closely fails to meet the lower threshold to be defined as interglacial. A slightly higher threshold leading to an interglacial here, in agreement with T17, would then also classify the period around MIS 13c (550 kyr BP) as interglacial, which would then disagree with the previous classificationδ18O prior to 1.5 Myr BP in T17 and can be interpreted as a change in the background climate by, e.g., a long-term change in CO2 concentration. However, there are still seven periods that have been identified as interglacials in T17 and that do not show up as interglacials when our criterion is applied  interglacial return times would be 60 (±22) kyr, 47 (±19) kyr, and 79 (±24) kyr for the pre-MPT, MPT, and post-MPT, respectively. However, similarly as the average 100-kyr periodicity in the post-MPT, that according to the idea of an obliquity-driven system with skipped termination causing either a  ~80 or  ~120 kyr return time of interglacials20, these mean values here also suggest some misleading average periodicities. A more helpful way seems to be to count the realized new interglacials with respect to the potential new interglacials, with the later being identical to the obliquity cycles. Following this idea we find a new interglacial (or termination) realization fraction of 67% in the pre-MPT , of 88% in the MPT , and of 52% in the post-MPT . These fractions should not be mixed with the fraction of obliquity cycles that include interglacials, since the realization of new interglacials decreases not only for obliquity cycles with a skipped termination but also for those with a continued interglacial.Thus, when using the occurance of little land ice in the northern hemisphere outside of Greenland as criteria for interglacials, we find that not only during the last 1 Myr but also from 2.6 to 1.6 Myr BP prior to the MPT the occurence of new interglacials is rather irregular. In four periods before the MPT  has already been detected as one of the obliquity cycles of the last 2 Myr with a skipped termination29 of benthic δ18O showed that prior to the MPT only 20% of the long-term signal (41-kyr running mean) has been caused by sea level or ice volume (δ18Osw), rising to 50% during glacial maxima after the MPT, while the dominant part of the change in benthic δ18O has been caused by ocean temperature variations  transform into rather small anomalies in the northern hemispheric ice-volume changes outside of Greenland. Thus, the detrending of the LR04 δ18O applied in T17 is according to our results too imprecise to correct as suggested for the effects of deep ocean temperature and ice volume in Antarctica and Greenland  or skipped terminations  are in detail based on different criteria.Skipped terminations identified previously for the last 2 Myrδ18O stack with currently available information—does not follow a simple rule. Instead, our findings suggest that across various parts of the Quaternary terminations leading to new interglacials are more irregularly placed and not necessarily forced by obliquity changes as is often assumed. This conclusion is conditional to the definition of an interglacial as having little ice in the Northern Hemisphere outside Greenland. Thus, alternative definitions of interglacials (as reviewed previously27) might be necessary when applied to the early Quaternary, and potentially also to earlier periods during the Neogene. Actually, our definition of interglacials clearly illustrates a shift in the climate system, from an interglacial-dominated period in the early Quaternary to a glacial-dominated period in the late Quaternary, a pattern which has originally been suggested from results of a simplified vertically integrated sectorial ice model without thermodynamics28. This becomes especially clear when we base our findings on constant thresholds  obliquity cycles without the onset of a new interglacial suggests that our classification—based on a deconvolution of the LR04 9 was forced solely by known changes in the orbital parameters and some assumptions on long-term changes in atmospheric CO2. The authors were able to simulate climate dynamics across the MPT confirming a dominant role of regolith removal on glacial dynamics, as proposed earlier15. The hypotheses of pressure-melting point dynamics in North American land ice as obtained from the ice-sheet distribution underlying this study here6 and of regolith removal15 as an explanation for the MPT are not mutually exclusive. They all point to ice-sheet dynamics as a dominant process responsible for the lengthening of the glacial cycles after the MPT, but they disagree in detail on how this is achieved.Most recently, an intermediate complex Earth system modelδ18O stack4. Decomposing the δ18O signal in temperature and ice volume showed29 that this trend is mainly based on a change in ice volume over the MPT rather than on a change in the temperature. This interpretation of little long-term change in the temperature of glacial maxima across the MPT agrees with a reconstruction31 of ocean temperature and sea level, but disagrees with another study32, which mainly found a decrease in the glacial temperature across the MPT.Climate change during the Quaternary consists not only of glacial/interglacial transitions, but contains also a long-term trend towards more positive values in the LR04 benthic 2 concentrations12 , which represents a relative uncertainty of 2%.As stated earlier, we understand our land ice distribution as a model-based interpretation of the LR04 benthic 45, the proxy- and model-based knowledge on northern hemisphere land ice through the Quaternary has been combined, highlighting also large gaps in our knowledge on times earlier in the Quaternary. However, the feature46 of a so far unexplained47 early Pleistocene glacial advance in North America as far south as 38° N dated around 2.5 Myr BP is not yet met by our results and cannot be reconciled with our present knowledge on climate change. Either we are missing some details on radiative forcing that has been active in the early Quaternary, or these data need to be reinterpreted, or ice sheets models used for simulating these events have been too simplistic. Note that in the context of our study, especially the minimum in land ice volume related to interglacials is of interest, which is even more weakly constrained by data than the maximum glacial extent. Furthermore, these findings46 suggest, that the existence of regolith might have promoted the existence of widespread North American ice sheets in the early Quaternary, but the absence of similar glacial advances for the following 1 Myr implies the absence of regolith thereafter. Those implications make these empirical data46 difficult to reconcile with the regolith removal hypothesis8.Reconstruction-based evidence on ice-sheet extents for these periods is rare. In a recent compilation29 of the LR04 δ18O stack might alternatively be used to evaluate of our results. Thus, sea level and δ18O of seawater (δ18Osw) of different approaches49 have been compiled —one of the dominant processes changing climate across the MPT. This information can be used to evaluate how important the choice of the latitude at which changes in orbital insolation are investigated in simplified approaches24 actually is. Interestingly, it turns out that although land ice before the MPT is mainly located north of 65°N, a precise knowledge of incoming solar energy (and therefore of the investigated latitude) is of secondary importance for the calculation of land ice albedo feedbacks. In other words, for the energy budget changes in land ice distribution and not incoming insolation  are most important. This implies that any reasonable latitude at which one finds northern hemispheric land ice  is a valid choice at which insolation changes are analyzed justifying in retrospective the idea for simple rules based on incoming insolation at 65°N.The latitudinal integration of land ice volume distribution across the Quaternary Fig. b clearlyδ18O stack. This raises the question if any such strict definition of interglacials  is a meaningful exercise. We propose, alternatively, that the gradual change of climate should be more in the focus of further research. Normalized time series of LR04 δ18O, the detrended δ18O used in T17 and of our northern hemispheric ice volume outside of Greenland clearly differ early in the Quaternary, while they converge to similar dynamics later on  would still hold, even when looking on these gradual changes that avoid any thresholds.Focusing the analysis of the Quaternary on the definition of interglacials reduces the variety of climate change to a binary point of view: a time period is either a glacial or an interglacial. The comparison of our results of the definition of interglacials with those obtained by T17 clearly showed that especially for times earlier in the Quaternary ambiguity exists, and the outcome of any such binary definition depends to a large extent on the chosen thresholds, their uncertainties and the individual records involved in the analysis, e.g., the benthic LR04  on Fig. . Any binδ18O stack and might therefore in some details be method dependent. However, our general finding, independent of uncertainties and approaches, is the following: The glacial/interglacial amplitudes of northern hemisphere land ice variability outside of Greenland are much smaller prior to than after the MPT. In the corresponding benthic δ18O stack this change in glacial/interglacial amplitude across the MPT is much weaker, since δ18O also contains contributions from deep ocean temperature and ice volume in Antarctica and Greenland. While regular obliquity-driven changes in benthic δ18O certainly appear prior to the MPT, our findings challenge if minima in benthic δ18O should be classified as interglacials in the traditional sense used so far in the literature, since related amplitudes in northern hemispheric land ice outside of Greenland are rather small before the MPT. Consequently, our approach would lead to a pattern of climate change during the Quaternary that might consist of irregularly appearing interglacials both prior to and after the MPT. While the latter has been proposed before by various studies due to skipped terminations24, the proposed irregular pattern prior to the MPT is, to our knowledge, new. Hence, the so-called 41-kyr world prior to the MPT would not be a period with regularly appearing glacial terminations , but be irregularly glaciated and deglaciated similar to the so-called 100-kyr world after the MPT. In detail, the fraction of realized new interglacials or terminations per obliquity cycle is 67%, 88% and 52% during the early, mid, and late Quaternary, respectively. Alternatively, we have to accept that the definition of interglacials might not be applicable straightforward to the whole Quaternary since this conclusion is solely based on our applied definition of interglacials—the absence of significant northern hemispheric land ice outside of Greenland, which has been suggested27 to be suitable for at least the last 800 kyr, and which agrees with other approaches24 for the last 1.6 Myr. Following our definitions the appearance of interglacials cleary shifts across the Quaternary, from an interglacial-dominated to glacial-dominated climate. Furthermore, any scheme that tries to define glacial and interglacial periods depends not only on the chosen variable for classification, but also largely on chosen thresholds and their uncertainities. Such a binary view on climate introduces unnecessary ambiguity in the analysis. We propose that less importance should be placed on such classifications, and more efforts should be made in understanding and improving the underlying physical mechanisms driving the climate changes as recorded in the geological data, including at least carbon cycle feedbacks and changes in land ice.Our analysis is based on only one model-based interpretation of a benthic 29 consisting of an updated version of an earlier study6. While the initial study6 was restricted to land ice dynamics in the northern hemisphere, the updated study now also simulates land ice in Antarctica. Within a modelling approach, the 3-D ice-sheet model ANICE is applied to deconvolve the benthic δ18O stack LR044 in deep-ocean temperature, global mean sea-level variations, and a representation of the four main ice sheets in Antarctica, Greenland, Eurasia, and North America. In detail, the modelling framework is in a forward mode forced to match the temporal changes in reconstructed benthic δ18O by physically consistent contributions from both deep ocean temperature change and land ice-volume (sea level) change. Areal extent and ice thickness of ice sheets and ice shelves from the four individually simulated ice masses in North America, Eurasia, Greenland, and Antarctica are available at their simulated spatial resolution of 40 × 40 km2 (Greenland at 20 × 20 km2). Results29 cover the last 5 Myr, but we restrict our analysis to the Quaternary as done in T17. Temporal resolution of the available spatially resolved land ice changes is 2 kyr. For the detection of the onset of interglacials (see below), these data are interpolated to 1 kyr. This rather coarse temporal resolution of the ice-sheet model results, but also of the underlying benthic δ18O stack LR04, does not allow us to test if a decrease in millennial-scale variability may have caused the lengthening of ice ages across the MPT, as suggested previously26.We use land ice simulation output27 and to minimize the amount of obliquity cycles without interglacials earlier in time. Threshold 1 is the value below which the ice volume needs to fall to detect an interglacial, threshold 2 is the value above which the ice volume needs to rise to separate two interglacials from each other. If threshold 1 is not crossed within an obliquity cycle, this cycle is called to contain a skipped termination . If threshold 2 is not crossed, the obliquity cycles contains a continued interglacial. In our standard case, the constraints given by the data and our goal to minimize the amount of obliquity cycles without interglacials lead to temporal changes in the chosen thresholds. For these, we use as additional boundary condition, that the difference between both thresholds stays constant over time. Being aware of uncertainties in the land ice volume of North American and Eurasian ice sheets, which have been estimated to 0.5 × 1015 m3 (1σ) in the model-based deconvolution of the LR04 benthic δ18O stack29, we choose threshold values only in full digits of ni × 1015 m3. In our standard case n1 is 7 and n2 10 after 1650 kyr BP, and 3 and 6 before 1750 kyr BP, respectively, with linear interpolation inbetween. In the alternative definitions, the thresholds are kept constant over time, using either those values determined for the early or late Quaternary (Fig. The thresholds used to define interglacials have been chosen in a way to find similar results for the last 800 kyr as in a recent review paper2 plotted in Fig. 532 values are close to those obtained with a revised approach54, as has been shown elsewhere55.The radiative forcing of CO56.Frequency analyses have been performed with AnalyseriesPeer Review File


The recent identification of the bio-duck call as Antarctic minke whale (AMW) vocalization allows the use of passive acoustic monitoring to retrospectively investigate year-round spatial-temporal patterns in minke whale occurrence in ice-covered areas. Here, we present an analysis of AMW occurrence patterns based on a 9-year passive acoustic dataset (2008–2016) from 21 locations throughout the Atlantic sector of the Southern Ocean (Weddell Sea). AMWs were detected acoustically at all mooring locations from May to December, with the highest presence between August and November . At the southernmost recording locations, the bio-duck call was present up to 10 months of the year. Substantial inter-annual variation in the seasonality of vocal activity correlated to variation in local ice concentration. Our analysis indicates that part of the AMW population stays in the Weddell Sea during austral winter. The period with the highest acoustic presence in the Weddell Sea (September–October) coincides with the timing of the breeding season of AMW in lower latitudes. The bio-duck call could therefore play a role in mating, although other behavioural functions of the call cannot be excluded to date. AMWs have a circum-Antarctic distribution and are known to occur within the marginal ice zone (MIZ) and the interior sea-ice pack [The Antarctic sea-ice environment constitutes a productive and dynamic habitat for many unique migratory and resident species, ranging from zooplankton, fish and birds to marine mammals . During ngthened ,14–17. Tice pack –20. Mostice pack . As a coice pack . Only veice pack –25.The recent identification of the bio-duck calls as vocalizations produced by AMWs  allows tThe identification of AMW sounds marked the end of a long-standing riddle as the bio-duck calls had been recorded at numerous locations across the Southern Ocean as well as off the Australian and Namibian coasts ,29–31. WHere, we use multi-year passive acoustic data from a recording network in the Weddell Sea and along the Greenwich meridian to investigate spatial-temporal patterns in the daily acoustic presence of AMWs.2.2.1.Passive acoustic data were collected between 2008 and 2016 (9 years) from 21 mooring positions  . For thiThe positions located in the Weddell Sea were sub-divided in four different sectors: Elephant Island (EI), Weddell Sea North (WSN), Weddell Sea South (WSS) and Greenwich Meridian (GW) . This su2.2.Triton [The acoustic recordings were processed using the Matlab-based  custom software program Triton  to creatTriton . For allWe used the bio-duck call as proxy for AMW acoustic presence  were used.2.3.r) to 18.1 * log10 (r). Noise levels at this position averaged 74.4 dB ± 4.2 dB. For identification of calls during the manual screening of spectrograms, it was assumed that the SNR of AMW vocalizations would need to exceed 3 dB. Resulting distance estimates for an AMW call with a source level (SL) = 147 dB re 1 µPa [The sea-ice concentration data used for this study were extracted from satellite images with a resolution of 6.25 × 6.25 km from the Advanced Microwave Scanning Radiometer for EOS (AMSR-E) satellite sensor . The radre 1 µPa , ranged re 1 µPa –46) and r = 0.89)To explore how AMW acoustic presence related to sea-ice concentration, ice concentration and acoustic presence data were correlated for every location using a Pearson's correlation. The variability of this relationship with the temporal resolution was explored by correlating AMW presence and sea-ice concentration within moving windows of varying temporal width, i.e. 1, 3, 7, 15, 21, 30 days, to select the best temporal resolution to correlate the acoustic presences with the ice concentration values. Monthly sea-ice concentration data showed highest correlation values . AMWs were found to be present at all except the northernmost position (GW5) in this study .Table 2For two positions (WA3 and WA4), the moorings each contained three recording devices (SonoVaults), which all were programmed to record simultaneously at different depths. Due to the fact that at these positions, all recorders dropped out for some part of the year and we compiled the data from all three recorders for each mooring to obtain one effective year for data analyses on AMW daily presence.3.1.AMW acoustic presence differed by sectors; at the southernmost positions, AMWs were generally acoustically present longer (i.e. over more months) than at the northern positions . At the AMW bio-duck occurrence exhibited in general a strong seasonal pattern, although AMW acoustic detections varied with location . In JanuIn the central Weddell Sea (WSN and WSS), the seasonality of the AMW acoustic presence did not exhibit a clear latitudinal gradient as in the GW sector. At some sites, the onset of acoustic presence was in June whereas at others, AMWs were acoustically present throughout 11 months. For all positions in the Weddell Sea, August, September and October generally exhibited the highest percentage of acoustic presence (greater than 80%) over the recording period (the only exception was position WA1 in August with 42% of days with acoustic presence). The positions WA1, WA3, WA4, WA5, WA6 and WA7 located between 63° S and 66° S (WSN) exhibited more days with acoustic presence compared to the positions located in lower latitudes, WA8 and WA9 (WSS). The positions WA1 and WA3 were the only positions located in the Weddell Sea sector that exhibited AMW acoustic presence during January . The posThe position at Elephant Island (EI) had a similar seasonal pattern of AMW, but with fewer days with detections. The first calls were detected during the last days of July (only 2% of days with acoustic detections). August and September were the peak . In November, the acoustic presence decreased to just 1% presence.AMWs were acoustically detected over 9 months (GW4), 11 months (GW2) and 12 months along with the GW sector. The overall percentage of monthly acoustic presence at the northern positions was substantially lower compared to the southern positions .In the sector WSN, one position had an acoustic activity for 11 months (WA1), four positions had between six and seven months of acoustic presence and two positions (WA2 & WA5) had two to four months with acoustic activity. However, recorders moored in these two positions collected data for only eight and five months. In the WSS sector, AMWs were detected eight months in WA8 and four months in the position WA9, where data collection occurred only during seven months. For the mooring positions located in the middle of the Weddell Sea area, there was no apparent north–south gradient in AMW acoustic presence .In the EI position, AMW were detected during only four months and at a low rate in contrast with the other sectors .3.2.Two sites with multi-year data, GW1 and GW2, had similar trends in AMW acoustic presence across years . It grad3.3.r = 0.7). Days with few detections generally corresponded to days with lower (less than 20%) ice concentration. In general, the acoustic presence of AMW in the Weddell Sea increased with local sea-ice concentration. The positive correlation explains the higher number of days with the acoustic presence with increasing latitude, given that sea-ice is more persistent in the southern recording positions  are distributed. In the central Weddell Sea, cycles of pancake-ice formation developing into consolidated floes are thought to be the dominant processes by which the sea-ice forms, starting from the centre . By contThe acoustic observations of AMWs overall show a strong association between their acoustic presence and sea-ice concentration during austral winter. Our findings on AMW distribution thereby contrast with the information from visual surveys indicating that AMWs occur primarily within the ice at the sea-ice edge. Visual data collection is, however, by and large limited to the austral summer period and areas with low ice cover. Furthermore, differences in AMW behaviour (e.g. surfacing patterns) when in ice-covered areas may also affect their detectability by visual observers and hence skew distribution information. To further insights on potential seasonal differences in AMW habitat usage, further investigations, e.g. employing animal-borne satellite tags, are clearly needed.4.2.et al. [To date little is known on the function of AMW calling behaviour. Across all locations where AMWs have been recorded, acoustic activity is strongly seasonal, albeit with regional differences in the timing of peak calling. Off the Western Antarctic Peninsula, most AMW calls were detected between April and November with peak calling during July . In our et al.  reportedet al. . As alsoet al. , these oet al. .et al. [et al. [Megaptera novaeangliae) and grey whales (Eschrichtius robustus), which prefer near shore waters for breeding. They hypothesized this may result in differences in meeting probability between mature males and females compared to species that concentrate in traditional breeding areas in coastal waters. The AMW long calling bouts consisting of repetitive pulses could therefore serve to attract and find mating partners over longer distances. Nevertheless, further work e.g. employing animal-borne tags is needed and underway to further investigate the behavioural contexts in which AMW calls are produced .et al. [et al. [Balaenoptera acutorostrata), pregnant females may separate socially to avoid niche overlap or to avoid males by migrating to other areas. Furthermore, occasional sightings of young unweaned common minke whale calves strongly suggest some females calve in more northerly waters outside the breeding areas, possibly skipping migration to low latitudes to calve [To date it is unknown how AMW sound production is related to sex, age and reproductive status. Information on staggered and sex-segregated migration in AMWs, presents patchy and partly contrasting information; Kasamatsu et al.  reportedet al. ,57. Lact [et al.  suggesteto calve ,60.How these patterns relate to the austral winter situation in Antarctic waters and how calling behaviour relates to sex, age and reproductive status in Antarctic minke whales is not known. However, based on existing evidence, it is possible that the reproductive and conditional status of females determines their migratory strategy.Although not further quantified here, different bio-duck type calls were observed in our dataset, in accordance to the findings of previous studies that collected similar data in other areas ,29,31,414.3.Balaenoptera physalus), humpback and Antarctic blue whales (B. musculus intermedia); see Geijer et al. [et al. [et al. [Our data show that, as for many baleen whale populations , which are known to prey on the species [Adaptations to inhabiting heavy sea-ice environments may enable AMWs to exploit this habitat on a year-round basis, potentially limiting the need for migration to low latitude waters to the period when calves are born . The den species . Their s species ,24. Thes species ,69. Our  species ,71) is t species  for a re5.The strong relation between AMWs and sea-ice suggests that this species is likely to be sensitive to climate-induced changes to its sea-ice habitat over time. Passive acoustics provide a highly effective tool for monitoring this species during austral winter. Data presented here suggest that the migratory behaviour of this species is more complex than previously thought. Part of the population may undertake seasonal migrations while another part may remain in the ice, with a continuous presence in the Antarctic Southern Ocean during the year. Overall abundance estimates for AMWs are currently around 500 000, whereas earlier assessments estimated 720 000 animals  represen


Tau protein is subject to phosphorylation by multiple kinases at more than 80 different sites. Some of these sites are associated with tau pathology and neurodegeneration, but other sites are modified in normal tau as well as in pathological tau. Although phosphorylation of tau at residues in the microtubule-binding repeats is thought to reduce tau association with microtubules, the functional consequences of other sites are poorly understood. The AT8 antibody recognizes a complex phosphoepitope site on tau that is detectable in a healthy brain but significantly increased in Alzheimer’s disease (AD) and other tauopathies. Previous studies showed that phosphorylation of tau at the AT8 site leads to exposure of an N-terminal sequence that promotes activation of a protein phosphatase 1 (PP1)/glycogen synthase 3 (GSK3) signaling pathway, which inhibits kinesin-1-based anterograde fast axonal transport (FAT). This finding suggests that phosphorylation may control tau conformation and function. However, the AT8 includes three distinct phosphorylated amino acids that may be differentially phosphorylated in normal and disease conditions. To evaluate the effects of specific phosphorylation sites in the AT8 epitope, recombinant, pseudophosphorylated tau proteins were perfused into the isolated squid axoplasm preparation to determine their effects on axonal signaling pathways and FAT. Results from these studies suggest a mechanism where specific phosphorylation events differentially impact tau conformation, promoting activation of independent signaling pathways that differentially affect FAT. Implications of findings here to our understanding of tau function in health and disease conditions are discussed. Surprisingly, we found that pseudophosphorylation of selected residues sufficed for monomeric tau to inhibit either anterograde FAT or both anterograde and retrograde FAT. Additionally, we present evidence linking these effects to PAD exposure and activation of selected kinase pathways. Collectively, findings here are consistent with a mechanism linking residue-specific phosphorylation of tau, stabilization of unique conformations, and activation of selected phosphotransferase signaling pathways. Implications of these findings to disease and developmental-related roles of tau are discussed.The following antibodies were used for Western blots: KHC  was added to a final concentration of 0.4 mM to induce protein expression. After 3 h, bacteria pellets were collected by centrifugation at 8,000 g and resuspended in lysis buffer  with protease inhibitor cocktail (Sigma–Aldrich). Cells were sonicated on ice and tau was purified from cleared lysates with Talon Metal Affinity Resin as per the manufacturer’s instructions (Takara Bio). Eluted proteins were concentrated and buffer exchanged into 50 mM HEPES, 100 mM KCl using Amicon 30 kDa columns (Millipore).Recombinant tau proteins in Loligo pealii  as described  supplemented with 5 mM ATP and perfused into isolated squid axoplasm at final concentration of 2 μM. For TNT1 blocking experiments  in MOPS buffer and proteins were transferred to PVDF membrane (Bio-Rad) using Towbin buffer with 10% methanol. Membranes were dried for at least 1 h before being reactivated in methanol and blocked in 1% milk in TBS supplemented with 2 mM Sodium Orthovanadate and 10 mM Sodium Fluoride to block phosphatase activity present in milk. Primary antibodies were incubated overnight diluted in 1% BSA in TBS supplemented as before. Goat anti-mouse or goat anti-rabbit secondary antibodies (LiCor) were incubated for 1 h at RT and immunoblots visualized using a LiCor Odyssey Fc imaging system. LiCor ImageStudio Lite was used for quantitation of signal intensity.n values shown in Figures). For vesicle motility assays, the difference between the mean of velocity measurements obtained between 0–10 and 30–50 min after perfusion was calculated for both anterograde and retrograde FAT in each experiment. For biochemistry experiments, signal intensities of “sister” axoplasms were quantified as described in Section “Immunoblotting-Based Analysis of Kinase Activity in Squid”  to PAD exposure and activation of a downstream PP1-GSK3β signaling pathway. However, ample data exists showing that some tau phosphospecies are detectable in the normal and pathological brain including phosphorylation of some, but not all three S/T residues in the AT8 antibody epitope. For example, pS202 and pT205 tau are detected in cerebrospinal fluid of AD patients, with pT205 showing the highest correlation with disease progression  to glutamic acid, which mimics the negative charge effect associated with phosphorylation . FollowiNext, we set out to determine whether the effects elicited by hTau40-T205E and hTau40-S199E on FAT were impacted by phosphorylation of the adjacent S202 residue. As shown in Exposure of PAD in tau, an event associated with various pathological post-translational modifications, has been shown to trigger a PP1-GSK3β pathway that inhibits anterograde FAT  can be differentially phosphorylated either singly or in different combinations  led to a conformational change in tau that exposed a biologically active motif in the N-terminal of tau that is normally sequestered in dephosphorylated tau (Kanaan et al., Despite the proximity of the three phosphorylation sites analyzed here, we found that single phosphorylation at S199 or T205, but not at S202, produces monomeric tau that differentially affects FAT by activation of two distinct downstream signaling pathways. Specifically, hTau40-T205E inhibited anterograde FAT through a mechanism involving exposure of the N-terminal PAD and PP1/GSK3β activation, as seen with hTau40-AT8 in prior studies (Kanaan et al., In vitro, pseudophosphorylation of the AT8 epitope exposes the N-terminal whereas pseudophosphorylation at the C-terminal PHF1 epitope promotes exposure of the C-terminal (Jeganathan et al., Tau is generally classified as an intrinsically disordered protein. However, multiple studies have indicated that monomeric tau normally folds into a “paperclip” conformation with the N- and C-terminals near the central microtubule-binding domains (Jeganathan et al., Investigation of dually pseudophosphorylated hTau40 proteins indicated additional levels of complexity. Both hTau40-S199E/S202E and hTau40-S199E similarly inhibited both directions of FAT, implying that S202 pseudophosphorylation does not alter the specific hTau40 conformation elicited by S199 pseudophosphorylation significantly. In contrast, pseudophosphorylation of S202 significantly changed the effects of hTau40-T205E. Whereas hTau40-T205E affected only anterograde FAT and the effect was blocked by TNT1, hTau40-S202E/T205E inhibited retrograde FAT and exhibited a slow decline in anterograde FAT that did not reach significance in 50 min. Co-perfusion with TNT1 did not change the effects on either anterograde or retrograde FAT by hTau40-S202E/T205E. Tau is known to have a dynamic secondary structure, which can be stabilized by phosphorylation of selected residues (Jeganathan et al., Although most studies on tau function have focused on microtubule dynamics, there is evidence that tau can also affect neuronal signaling in other ways. For example, tau has also been suggested to potentiate NGF-ERK signal transduction in differentiating PC12 cells specifically through a mechanism involving phosphorylation at T231. Tau depleted cells were unable to initiate neurite extension and the effect could be rescued by exogenous expression of WT but not Tau-T231A (Leugers and Lee, While tau phosphorylation has commonly been studied in the context of disease, various lines of evidence indicate that it also has additional physiological functions, especially important during development. The relative levels of tau phosphorylation and the specific sits modified change during the development and maturation of the brain. When tau from the fetal brain is analyzed for the amount of phosphorylation, it has approximately six phosphates per molecule of tau whereas in tau from the adult human brain the ratio is 2–3 mol of phosphate per mol of protein (Kenessey and Yen, Further evidence of this is seen from the enrichment of AT8 immunoreactivity in gray matter, which is typically at low levels in adulthood, but at higher levels during development and in tauopathies (Brion et al., in vitro (Despres et al., It is important to note that a fourth phosphorylation site in tau, pS208, may also be recognized by the AT8 antibody (Malia et al., In vitro S199, S202, T205 all have the potential to be phosphorylated by at least seven kinases, including GSK3β and Cdk5 (Singh et al., in vivo, tau certainly has the potential to be a central point for multiple signaling pathways potentiating disparate downstream functions, depending on which site is modified. Equally important is switching off the signaling by dephosphorylation. The major phosphatase for tau is PP2A accounting for ~70% of the activity, but certain sites are preferentially dephosphorylated by either PP2B or PP1 (Liu et al., Phosphoprotein-species are maintained by an intricate balance of kinase and phosphatase activities, which are spatially and temporally regulated. Many questions remain about the role of tau in spatial and temporal regulation of signaling pathways, suggesting this is a rich area for further study. For example, an important feature of tau biology not addressed here relates to isoform-specific differences (Cox et al., The data presented in this study strongly supports our hypothesis that phosphorylation of tau at individual sites can modulate tau function as a signaling hub. Tau is well known to have a highly dynamic protein structure and several lines of research suggest that phosphorylation of tau at specific sites can stabilize distinct conformations. These conformations may allow activation of diverse signaling pathways through the exposure of multiple biologically active domains such as the N-terminal PAD and others yet to be defined (Ittner et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because IACUC approval was not required for this study. All recommendations for humane use of squid set by the Marine Biological Laboratory were followed.SM and SB conceived and designed the study, plotted and analyzed the data. M-YT expressed and purified the recombinant proteins. KB, SA, SK, and SM collected the data with additional experimental assistance from GM and SB. SM, GM, and SB wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The “taciturn man from the North” is how his contemporaries described Caspar David Friedrich, referring to his melancholy, or in his own words, his “dreadful weariness,” especially in later years. Loneliness pervaded his work as well as his life, which was marred by early deaths in the family―of his mother when he was seven and several siblings, among them, a young brother, who drowned in a frozen lake, according to some, trying to rescue him.Friedrich was born in Greifswald, then Swedish Pomerania, on the Baltic coast of Germany, the son of a candle maker and soap boiler in a family of 10 children. As a youth he studied with architect and painter Johann Gottfried Quistorp but later moved to Copenhagen to attend the Academy, one of the leading centers of art in Europe, and eventually settled in Dresden. His training in the neoclassical tradition relied on extensive preliminary studies, drawings, and sketches to depict the physical world and is reflected in the disciplined quality of all his works. But while his landscapes were always actual studies of nature, they were more than a representation of nature.This man, who according to his contemporaries discovered “the tragedy of landscape” and gained by it fame in his own time, soon embraced an untested individual approach to painting, despite a lingering attachment to the systematic techniques of his training. “The artist should paint not only what he sees before him, but also what he sees within him,” he wrote. This belief was rooted in his view of nature as a subject itself worthy of study, imbued with spiritual qualities and portrayed entirely without human presence, not as backdrop but as protagonist. His interest was not in the beauty of nature alone but in what the romantics called the sublime―powerful natural phenomena: snowstorms, impenetrable fog, impassable mountains―generating conflicted feelings of wonder and helplessness, which he could sense and capture with symbols and allegorical elements.Viewing and presenting the landscape in an entirely new way was Friedrich’s main innovation. He turned the mountains, forests, and vistas of northern German countryside in the times of Beethoven, Schubert, and Goethe into romantic icons, painting them at all times of night and day, around Dresden and the River Elbe, especially in the moonlight and sunlight or covered with mist. “Close your bodily eye so that you may see your picture first with the spiritual eye. Then bring to the light of day that which you have seen in the darkness so that it may react upon others from the outside inwards,” he wrote in his notes on aesthetics in 1830. Therefore, his winter landscapes were not about life in the winter but about winter itself, stark, still, desolate, where “no man has yet set his foot.”Despite early fame and a prolific career, Friedrich lost ground in his mature years and fell into poverty, becoming the “most solitary of the solitary.” Bare trees and stumps populated with ravens and owls near graveyards and ruins filled his works, expressing the passage of time and his own state of mind. But these late paintings also explored a mystical approach, one abandoning the self to reach an intuitive understanding of physical phenomena. This period’s frisson of the sublime was later adopted by Hollywood directors to show horror, trepidation, and other emotions caused by human inadequacy against the overpowering forces of nature.The Polar Sea, on this month’s cover, expresses Friedrich’s mature vision, which, far ahead of his times, was not well received. The painting was inspired by William Parry’s arctic expedition of 1819–20, a venture filled with opportunities for symbolic interpretation. The artist seized these to build a monument to nature’s triumph over human efforts to conquer it. The tiny image of the ship, inscribed HMS Griber, against a mount of ice, signals the insignificance of human enterprise. Frightful shards jut into the steel gray sky atop solid slabs of ice that form a frigid grave over what human presence might have existed before the wreck and builds a wall between the viewer and the ship.Another leading romantic, Samuel Taylor Coleridge wrote prolifically about imagery deep with symbolism. In “The Rime of the Ancient Mariner,” he offered his version of beautiful and ominous nature, set in a metaphysical world. Among the many influences on this poem, were vivid accounts by arctic explorers. Like Friedrich, Coleridge was fascinated by their travails, which he immortalized. Here is the Mariner’s ship in the grip of polar ice: “And now there came both mist and snow, / And it grew wondrous cold: / The ice, mast high, came floating by / As green as emerald…. / The ice, was here, the ice was there, / the ice was all around: / It cracked and growled, and roared and howled, / Like noises in a swound!/”The “rime” in the world of both Friedrich and Coleridge is symbolic of the sublime world of nature. At once fascinating and terrifying, it changes forms: water, ice, mist―taxing visual awareness, toying with the artist, challenging the scientist, tempting the poet. When European mariners were searching for the Northwest Passage, formidable polar ice lay between them and navigation. The routing was lined with myth and uncertainty, hunger, and scurvy. How times have changed! Now instead of the powerful solidity of ice, we fear instead its fragility as the polar ice cap threatens to melt into the sea, exposing among other puzzles, the dynamic evolutionary interface between human viruses and the ice that can preserve and protect them for thousands of years. What remains constant is nature’s upper hand.Forsete arrived at Longyearbyen, a tiny village in Spitsbergen Island, Svalbard, Norway, north of the Arctic Circle. An outbreak of flu had broken out on the ship caused as it turned out by an extraordinarily potent strain that would become known as Spanish Flu. Many passengers, young miners, were hospitalized and over the next few weeks, seven of them died. Their bodies, containing the deadliest flu virus the world has ever known, were buried in the local cemetery, 800 miles from the North Pole.In 1918, as explorers were plowing their way into the Arctic, other events were also making history. World War I was coming to a close, yet weary humanity already had a new serious concern, one that was to cause more deaths around the globe than this and future wars combined. The public health emergency spread widely in the fall of the year. Only 3 days after taking sail, the Almost 8 decades later, a similar grave in Alaska permafrost held valuable clues about the Spanish Flu pandemic. Unlike the one concocted in Friedrich’s imagination, this grave was not a monument to human failure. Its contents enabled RNA sequencing of much of the 1918 virus. As the ice melts, more secrets of the great pandemic may see the light of day, guiding present flu prevention activities. Moreover, other illnesses become endemic in new areas as a result of changes in climate. Tick-borne encephalitis seems to be moving northwards in Europe and shifting upwards on 84 mountains apparently influenced by such changes. Frozen solid or melting fast, sublime nature rules.


The use of laparoscopic surgery has become widespread in recent years. One of its complications is port site hernia (PHS). It can be difficult to close the fascia at the time of laparoscopy, especially in obese patients, and there is a risk of herniation through a fascial defect with incomplete closure. It is important to ascertain closure of the defect when repairing PHS to prevent recurrence. We report a 47-year-old woman who developed a PHS at the superior aspect of the umbilicus. We repaired the defect using the VersaOneTM Fascial Closure System with laparoscopic guidance. This system allows the port site to be reliably closed while observing the suture from the abdominal cavity. The incision is the same size as a port site. If the abdominal wall is thick and the PHS has a diameter of ~10 mm, this method is considered to be indicated, regardless of the site. As laparoscopic surgery has become an increasingly common procedure, reports of port site hernias (PHS) have been increasing. Although the laparoscopic approach creates smaller incisions than laparotomy, it is more difficult to close the fascia, especially when the abdominal wall is thick. There is a risk for future herniation through any remaining fascial defect. Repairing PHS requires certainty of complete fascial closure to prevent recurrence. We repair PHS using the VersaOne™ Fascial Closure System with laparoscopic guidance, with good results.2. Her medical history included uterine fibroids and ovarian cysts. She had no history of smoking. Magnetic resonance imaging (MRI) revealed protrusion of adipose tissue at the upper side of the umbilical region, through a fascial defect measuring 9 × 13 mm; diastasis recti was also noted (A 47-year-old woman was referred to us by her obstetrician. She developed a PHS after laparoscopic total hysterectomy with right oophorectomy performed 3 years previously. She reported pain with intermittent bulging on the superior aspect of the umbilical region. Clinical examination revealed a single hernia orifice at that location. Her height was 161.1 cm, with a weight of 60.1 kg, yielding a body mass index of 23.16 kg/mso noted  and 2.Under general anesthesia, with the patient in the lithotomy position, a 5-mm trocar was inserted into the lower right abdomen using the optical method. Observation of the abdominal cavity revealed a hernia orifice with a diameter of ~1 cm at the upper aspect of the umbilicus . Two addThis system uses a 12-mm trocar, through which an inner cylinder can be inserted to use as a guide for suture closure. The guide contains two diagonal channels. As shown in We inserted the system’s 12-mm trocar into the hernia orifice  and inseThe patient did well after surgery. Follow-up MRI taken 10 days after surgery confirmed the absence of hernia and closure of the fascial defect  and 10.In recent years, the use of laparoscopic surgery has become widespread. Although PHS is relatively rare, occurring in 0.26–2.5% of patients who undergo laparoscopic surgery , its incWhen an abdominal incisional hernia occurs, the options for repair are suturing using native tissue, repair using mesh or a combination. The recurrence rate of simple suture closure in Japan is 9.3–11.4%, higher than the recurrence rate of mesh repair that is 2.4–4.3% . HoweverOur patient had a 9 × 13-mm fascial defect at a prior port site, which we were able to successfully repair using the VersaOne™ Fascial Closure System. It is indicated for patients with an abdominal wall thickness exceeding 30 mm, in whom reliable suturing of the port site is not easy. With this system, the port site can be reliably closed, including both fascia and peritoneum, while observing the suture from the abdominal cavity with a laparoscope. Because PHS can reportedly develop below the fascia , closureWith the closure system, the peritoneum at the hernia site is dissected from an intra-abdominal approach, and a skin incision is made at the same site for trocar placement. Since the peritoneum has been dissected in advance, the trocar can be easily placed. The incision is the size of a port: much smaller and less noticeable than the incision required for a normal hernia repair, conferring an aesthetic advantage.Closure of PHS defects is made more reliable by the use of a Z suture. For example, for a hernia with a diameter of 10 mm, the length inside the hernia is ~3 cm, and the suture length when simply doing knotted suture is ~1.5 cm. The use of two separate knotted sutures or Z sutures may be more reliable for wounds of this length.To date, this method has been used in ~30 patients without complications or recurrences. In patients with a thick abdominal wall and a PHS about 10 mm in diameter, this system is indicated regardless of the site. It will be necessary to accumulate more cases and follow patients to determine the long-term prognosis after using this surgical technique.None declared.