N-methyl-d-aspartate receptors (NMDARs) are transmembrane proteins that are activated by the neurotransmitter glutamate and are found at most excitatory vertebrate synapses. NMDAR channel blockers, an antagonist class of broad pharmacological and clinical significance, inhibit by occluding the NMDAR ion channel. A vast literature demonstrates that NMDAR channel blockers, including MK-801, phencyclidine, ketamine, and the Alzheimer’s disease drug memantine, can bind and unbind only when the NMDAR channel is open. Here we use electrophysiological recordings from transfected tsA201 cells and cultured neurons, NMDAR structural modeling, and custom-synthesized compounds to show that NMDAR channel blockers can enter the channel through two routes: the well-known hydrophilic path from extracellular solution to channel through the open channel gate, and also a hydrophobic path from plasma membrane to channel through a gated fenestration (“membrane-to-channel inhibition” (MCI)). Our demonstration that ligand-gated channels are subject to MCI, as are voltage-gated channels, highlights the broad expression of this inhibitory mechanism. Wilcox et al. (2022) show that NMDA receptor channel blockers, some of which are clinically important drugs, can access their binding site via 2 routes: a well-known path from the extracellular solution, and another path through the plasma membrane. N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors (iGluRs) present at most excitatory synapses in mammalian brains. Among iGluRs, NMDARs exhibit unique features including voltage-dependent block by Mg2+, high permeability to Ca2+, and slow deactivation kinetics3. Ca2+ influx through NMDARs activates multiple intracellular signaling pathways involved in synaptic plasticity, and contributes to learning and memory2. Pathological NMDAR activation, however, is implicated in Alzheimer’s disease, schizophrenia, major depressive disorder, and many other neurological and neuropsychiatric disorders5.1. NMDAR inhibition has been extensively studied to understand receptor structure and function, and to develop improved therapeutics. NMDAR channel blockers are inhibitors that block current by binding to the “deep site” at the outer tips of the pore-lining M2 reentrant loops near the middle of the ion channel9. Channel blockers typically can access their binding site only when the channel is open, and thus are also referred to as open channel blockers. NMDAR channel blockers examined for therapeutic use include Mg2+, amantadine, ketamine, MK-801, dextrorphan, phencyclidine (PCP)14, and memantine. Memantine has found particular clinical success as an FDA-approved treatment for Alzheimer’s disease15, and is effective for treatment of numerous other pathological conditions17.NMDARs are heterotetramers typically composed of two GluN1 and two GluN2(A-D) and/or GluN3(A-B) subunits. NMDARs can assemble as diheteromers  or triheteromers 19. Memantine was found to bind with weak apparent membrane voltage (Vm) dependence to a second site that was hypothesized to be superficial to the channel gate and accessible when NMDARs are closed19 (but see ref. 20). It initially was assumed that occupation of the second site caused NMDAR inhibition. However, this assumption was demonstrated to be incorrect8. Second site inhibition was found to require at least 2 steps: memantine first occupies a second site without inhibiting NMDAR-mediated current, and then transits from the second site to the deep site, where inhibition occurs. This mechanism suggests that “second site inhibition” is a misnomer; occupation of the second site does not produce inhibition, but rather sequesters memantine in a location from which it subsequently can transit to the deep site. The nature and location of the second site has remained a mystery.Memantine inhibition has also been observed following exposure of NMDARs to memantine in the absence of agonist, producing “superficial site” or “second site” inhibition21, including voltage-gated Na+ channels (VGSCs)28 and possibly NMDARs33. Integrating results from NMDAR models, custom-synthesized blockers, and electrophysiological experiments, we conclude that uncharged memantine can enter the membrane and transit to the deep site upon NMDAR activation through gated, membrane-facing fenestrations. Based on our findings we renamed second site inhibition “membrane-to-channel inhibition” (MCI). Our results reveal that one of the most extensively studied classes of clinically significant neuroactive drugs can bind to NMDARs via either of two mechanisms.Here we test the hypothesis that the plasma membrane is the second site. Previous work suggested the plasma membrane is an important route through which lipophilic molecules can access membrane-associated proteins18 unless otherwise specified. The protocol consisted of the following steps  was measured; control extracellular solution was applied for 10 s to allow full NMDAR deactivation; memantine in 0 Glu was applied for 30 s; memantine was washed away by a 1 s application of control solution; 1 mM Glu was reapplied for 20–30 s and NMDAR-mediated current reflecting MCI and recovery from MCI (IMCI) was measured; control solution was applied for 41 s; 1 mM Glu was reapplied for 20 s and control NMDAR-mediated current after MCI (IControl2) was measured. Figure Control (IControl = (IControl1 + IControl2)/2) and of IMCI (middle); and a point-by-point ratio (Methods) of IMCI/IControl (right). The minimum value of the IMCI/IControl point-by-point ratio was measured .To quantify memantine MCI without contamination by “traditional” channel block (mediated by memantine entry into the open channel from the extracellular solution), the protocol shown in Fig. eps Fig. : 1 mM glred Fig. , right a7. A 1 s wash after memantine application was used because: it is brief enough to allow MCI measurement (time constant of recovery from memantine MCI is ~2 s (ref. 7)); it is long enough to completely eliminate memantine from the extracellular solution (Methods), ensuring that MCI measurements were not contaminated by traditional channel block.Memantine was applied for 30 s because longer applications did not increase inhibition8. Based in part on the Vm independence of memantine association with the second site, we proposed that the “second site” might represent a pool or reservoir of memantine in the plasma membrane8. Here we test that hypothesis.A key previous finding was that memantine associates with the “second site” equally well at +35 or at −65 mV34. Uncharged memantine is highly hydrophobic37 and thus resides predominantly in the membrane.Memantine is a primary amine in equilibrium between charged (protonated) and uncharged (unprotonated) forms Fig. . The chaa = 10.4; ref. 36). The fraction of uncharged memantine in aqueous solution increases as pH increases  by raising the pH from 7.2 to 9.0  was ~2-fold higher at pH 9.0  than pH 7.2 , consistent with previous blocker pH sensitivity measurements39, suggesting that charged memantine mediates traditional channel block.We first examined memantine MCI with all extracellular solutions at pH 9.0 Fig. , a pH at9.0 Fig. . In cont40 or pH-sensitive NMDAR properties43. We therefore designed pH jump protocols in which NMDAR activation occurred at pH 7.2, and pH was altered only during memantine application. The pH of the memantine-containing solution only was changed to pH 9.0 (“pH 9.0 jump”) to increase, or pH 6.3 (“pH 6.3 jump”) to decrease, the [uncharged memantine] . Similar to MCI experiments performed entirely at pH 9.0, the pH 9.0 jump experiments showed that MCI by 100 μM memantine was greatly augmented (Min IMCI/IControl reduced) relative to measurements at pH 7.2  because of low memantine MCI potency at pH 6.3, and only one parameter (IC50) was free during fitting (see Methods). We found that memantine MCI IC50 is powerfully pH dependent ), yielding a pKa of 10.7 for both memantine (in agreement with the measured pKa of 10.4 (ref. 36)) and DMM. Thus, the pH sensitivity of DMM MCI should be similar to that of memantine. We compared MCI by TMM and DMM at similar concentrations relative to their traditional IC50s. Observing robust memantine MCI at pH 7.2, where the memantine MCI IC50 is 71 μM . Because of the high traditional IC50s of TMM (72.3 μM) and DMM (16.8 μM) we were concerned that using 100-fold higher concentrations would lead to non-specific effects. We therefore performed experiments at constant pH 9.0, where memantine’s MCI IC50 is only moderately higher than its traditional IC50. At pH 9.0, 1 mM TMM  exhibited no MCI  displayed strong MCI  were similar . Whether MCI is exhibited by other channel blockers has not previously been examined.If the second site is the membrane rather than a true binding site, MCI should be exhibited relatively nonspecifically by NMDAR channel blockers with a titratable nitrogen. However, the path from membrane to channel may show some selectivity among channel blockers, as suggested by our previous observation that ketamine does not exhibit MCI47; Fig. 18. Figure To determine whether MCI is widely expressed among NMDAR channel blockers with a titratable nitrogen, we tested the NMDAR channel blockers PCP Fig. , MK-801 7) is that the second site is an external binding site on NMDARs from which channel blockers transit to the deep site during MCI. We performed two types of experiments to distinguish these alternative hypotheses.If the second site is the membrane, then MCI must result from transit of blocker from a “reservoir” of drug molecules within the membrane to the deep site. The principal alternative hypothesis . Because this measurement was made during simultaneous NMDAR activation and inhibition, the time constant of MCI onset is likely to be faster than 46.4 ms. Thus, memantine MCI onset is fast enough to partly inhibit NMDAR responses before current reaches its peak value, which, with the perfusion system and other experimental conditions used here, typically requires 30–40 ms.Although a small preinhibition peak was often visible with memantine  using extracellular [MK-801]s of 1 and 10 μM. At both [MK-801]s we observed a clear preinhibition peak followed by a time-dependent current decay  to induce nearly complete unbinding of memantine from the deep site51. Note that this protocol would not be feasible with MK-801 because its slow unbinding kinetics would require much longer depolarizations. If the second site represents specific binding site(s) for one or a few molecules, then after the depolarizing steps there should be no memantine left to transit to the deep site. In this case, memantine reblock following the depolarizations should be slight or nonexistent. However, if the second site represents a memantine reservoir (and time course of recovery from memantine MCI represents reservoir depletion), then blocker should transit continuously from membrane to deep site during recovery from MCI. In this case, reblock should be observed following each depolarizing step. We performed MCI protocols without or with two Vm steps imposed during recovery from MCI and measured IMCI/IControl just after the end of the second Vm step . A parsimonious explanation for these data is that the second site represents a reservoir of memantine in the plasma membrane.The second experiment used to distinguish the drug reservoir and the specific binding site hypotheses took advantage of previous measurements of the voltage dependence of the MCI process. Although occupation of the second site is voltage-independenttep Fig. . IMCI/ICeps Fig. , suggest52. We generated an open state structure using a steered molecular dynamics simulation protocol previously employed to model the AMPA receptor (AMPAR) TMD in the open state53. For Model 2 we started with a closed cryo-EM GluN1/2A NMDAR structure54 and generated an open state structure using a homomeric GluA2 AMPAR structure in the open state55 as a template. The NMDAR Model 2 open state structure in water and lipid is shown in Fig. + through the external gate of Model 2  through which channel blockers can pass, but only when NMDAR channels are open. To attempt to identify such a path we developed two separate open state models of the NMDAR TMD (Model 1 and Model 2). For Model 1 we started with a previously developed closed GluN1/2A receptor TMD model56. We found a single lipid to channel path (fenestration) unique to the open structure. The path is formed by channel opening mainly as a result of repositioning of the hydrophobic side chains of residues in the M3 and M1 helices of the GluN2A subunits  and GluN2A(M630W), mutations predicted by in silico mutagenesis , and GluN1/2A(M630W) NMDARs by recording currents during coapplication of memantine and glutamate , and GluN1/2A(M630W) NMDARs. Mutation GluN2A(M630A) decreased memantine MCI IC50  receptors than for WT receptors  exhibited by GluN1/2A(A570W) receptors could have resulted from increased memantine potency at the deep site rather than a specific effect on the MCI pathway.We also examined the effect on memantine MCI of single or double mutations involving other residues predicted to line the fenestration , not at the narrow constriction): GluN1/2A(A570W), GluN1/2A(I571W), GluN1/2A(M630W)(A570W) and GluN1/2A(M630W)(I571W)  and inhibit current. The two paths by which memantine, and other NMDAR blockers, can access the NMDAR deep site are illustrated in Fig. 8 that occupation of the “second site” (from which memantine transits to the deep site during MCI) is voltage independent. We examined the dependence of MCI on pH because the concentration of uncharged (deprotonated) memantine increases as pH increases. Manipulation of pH has been used previously to modulate the protonation state of local anesthetics during inhibition of VGSCs27. We found that memantine MCI potency increased dramatically as pH was raised, even when pH was modified only during memantine application . We found that the previously described channel blockers PCP, MK-801, dextrorphan, and RL-208 all exhibit MCI . We propose that inhibition of neuronal NMDARs by high intracellular MK-801 is a consequence of MCI.Uncharged memantine is strongly hydrophobic: the LogP of memantine is ~3MCI Fig. . InteresThe channel blockers examined here exhibited MCI with less potency than traditional channel block, although the relation between traditional channel block potency and MCI potency varied. Thus, the preferred route of access of the blockers we examined may be from the extracellular solution, as would be expected, since these blockers were identified as traditional channel blockers. However, because the MCI protocol requires a 1-s wash before inhibition is quantified, we may have underestimated blocker MCI potency, especially if there is a rapid component of blocker exit from the membrane. Further studies may reveal that the preferred route of access of some of the blockers tested here, or of yet untested channel blockers, is through the membrane.8. This observation provides compelling evidence that our MCI protocol , which for memantine is so fast that it could not be accurately resolved, is relatively slow for MK-801. We found a highly significant dependence of τM on [MK-801]  should result in faster MCI onset. We used MK-801 for this test because the time constant of MCI onset . We identified a residue (GluN2AM630)) that forms a constriction in the fenestration observed in open channels Fig. . In sili30) that 28, and permanently charged local anesthetics are unable to act on VGSCs through the hydrophobic path22. Pore access from the membrane through fenestrations also occurs in voltage-gated K+ channels67 and voltage-gated Ca2+ channels70. Several iGluR ligands were previously proposed to act at hydrophobic sites with unknown properties, including: the NMDAR channel blockers MK-80129 and ketamine30; the NMDAR inhibitor and local anesthetic bupivacaine;32 cholesterol, which is required for NMDAR function;31 the AMPAR inhibitor fluoxetine57. In addition, ligands for many other membrane proteins have been proposed to depend on partition into the plasma membrane, including antidepressants71, cannabinoids72, sphingosine 1-phosphate receptor ligands73, and β2-adrenertic receptor agonists74.Receptor modulation of other ion channels through lipophilic pathways is well established. Local anesthetics can access their binding site in VGSCs via hydrophobic fenestrationsThus, regulation of membrane proteins by ligands that travel though the plasma membrane is widespread. We expect that modulation of ionotropic glutamate receptors through hydrophobic pathways, as exemplified by MCI of NMDARs, will be found to be a mechanism of broad significance.75 in DMEM supplemented with 10% fetal bovine serum and 1% GlutaMAX (Thermo Fisher Scientific). 1 × 105 cells/dish were plated on 15 mm glass coverslips treated with poly D-lysine (0.1 mg/ml) and rat-tail collagen  in 35 mm petri dishes.Experiments were performed on the tsA201 cell line , a variant of the HEK 293 cell line, and on primary cultures of rat cortical neurons. tsA201 cells were maintainedWildtype or mutant GluN1/2A receptors were used for all tsA201 cell experiments. 12–24 h after plating, tsA201 cells were transiently co-transfected using FuGENE6 Transfection Reagent (Promega) with mammalian expression plasmids that contained cDNAs encoding enhanced green fluorescent protein (EGFP in pRK7) for identification of transfected cells, the rat GluN1-1a subunit , and the rat GluN2A subunit (GenBank M91561 in pcDNA1). For some experiments cells were transfected with GluN1 and EGFP:pIRES:GluN2A ), which was constructed by inserting EGFP in pIRES (Clontech) under transcriptional control of the CMV promoter, and rat GluN2A cDNA (GenBank D13211) after the IRES sequence. Voltage clamp recordings were performed on tsA201 cells 12–48 h after transfection.d,l-2-amino-5-phosphonopentanoate (200 μM) was added to the culture medium to prevent NMDAR-mediated cell death.Site-directed mutagenesis was performed on cDNAs encoding GluN1 and GluN2A subunit genes in ampicillin resistance-encoding plasmids (pcDNA 3.1 or pcDNA1) using the Stratagene Quik-Change XL sited-directed mutagenesis kit. Mutagenized NMDAR subunit cDNAs from isolated colonies were sequenced from 100–200 bases upstream to 100–200 bases downstream of each mutation (University of Pittsburgh Genomics and Proteomics Core Laboratories). cDNA ratios used in transfection were 1:1:1  or 1:1 (GluN1 and EGFP:pIRES:GluN2A). Following transfection, the competitive NMDAR antagonist 76 following procedures approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Embryos were removed from pregnant rats sacrificed by CO2 inhalation, embryonic cortical cells were dissociated using trypsin, and were plated on 12-mm glass coverslips  in six-well plates. Cell proliferation was inhibited after 2 weeks with 1–2 μM cytosine arabinoside. Recordings were performed on cultured neurons between 19 and 25 days in vitro.Primary cultures of cortical rat neurons were prepared from day 16 Sprague-Dawley rat embryos2, 10 HEPES, 0.01 EDTA. pH was balanced to 7.2 ±  0.05 or 9.0 ±  0.1 with NaOH, or to 6.3 ±  0.05 with HCl. Osmolality was raised to 290 ± 10 mOsm with sucrose. For experiments with tsA201 cells, all extracellular solutions contained 0.1 mM of the NMDAR agonist glycine. For experiments with neurons in primary culture, all extracellular solutions contained 10 μM glycine, 1 μM tetrodotoxin to prevent action potential generation, and 1 μM Ro 25-6981 to inhibit GluN1/2B receptors. Intracellular (pipette) solution contained (in mM): 130 CsCl, 10 HEPES, 10 BAPTA, and 4 MgATP. pH was 7.2 ± 0.05 with CsOH. Osmolality was adjusted to 280 ± 10 mOsm.The control extracellular bath solution contained (in mM): 140 NaCl, 2.8 KCl, 1 CaCla ~7.5), we did not change pH buffer to avoid simultaneously changing two conditions (pH and pH buffer). To test how well pH was maintained in our experiments we prepared the pH 9.0 extracellular solution and left it at room temperature for 3 h ; the solution was loaded into the fast perfusion system reservoirs; solution was allowed to flow for 25 min ; a solution sample then was collected and its pH measured. The measured pH (8.89 ± 0.03 (n = 3)) suggested that pH was adequately maintained in our experiments.Although pH 6.3 and pH 9.0 are outside the useful buffering range of HEPES (pKd-APV (Hello Bio), phencyclidine (Sigma-Aldrich), MK-801 (Hello Bio), dextromethorphan (Sigma-Aldrich), RL-208 , NMDA (Tocris), tetrodotoxin (Abcam), Ro 25-6981 (Tocris), and amiloride hydrochloride (Tocris).Drugs and their sources were: memantine (Tocris), trimethyl memantine and dimethyl memantine (see “Synthesis and purification of TMM and DMM” below), m of −65 mV  unless otherwise indicated. Whole-cell currents were recorded using an Axopatch 200B patch-clamp amplifier (Molecular Devices). Series resistance was compensated 80–90% using the prediction and correction circuitry. Currents were low-pass filtered at 5 kHz and sampled with a Digidata 1440 A at 10 or 20 kHz in pClamp10 (Molecular Devices). Current traces for presentation were refiltered offline in Clampfit 10.7 at 50 Hz. NMDAR responses were activated by fast perfusion of 1 mM glutamate (tsA201 cells) or 100 μM NMDA (cultured neurons).Pipettes were pulled from borosilicate capillary tubing (Sutter Instruments) on a Flaming Brown P-97 microelectrode puller (Sutter Instruments) and polished with a heated filament to a resistance of 2–5 MΩ. Whole-cell recordings were made from cells expressing eGFP identified by epifluorescence illumination on an inverted Zeiss Axiovert microscope. Cells were held at a V77. A critical requirement of our fast perfusion system was that the 1 s wash between application of channel blocker and application of 1 mM glutamate  we demonstrated previously using our fast perfusion system that no NMDAR response inhibition was observed after: (a) a 1 s wash was used8 to eliminate 50 μM D-APV (~200-fold above the D-APV Ki78); (b) after a 1 s wash was used18 to eliminate 1.4 mM Mg2+ (~250-fold above the Mg2+ IC50 at −65 mV79); (c) when a 0.4 s wash was used7 to eliminate 500 μM ketamine . The ketamine experiment provides a particularly stringent demonstration that our MCI measurements are not contaminated by inhibition due to traditional channel block.Solutions were delivered to cells using a ten-barrel fast perfusion systemsee Fig.  effectiv− may have hidden MCI by TMM, we repeated the MCI protocol shown in Fig. + + 1 mM I− in solution) replacing 1 mM TMM (1 mM TMM+ + 1 mM I− in solution). We found no difference between Min IMCI/IControl measurements with 1 mM NaI (n = 4) and with 1 mM TMM , suggesting that the presence of I− did not prevent observation of MCI by TMM.The iodide salt of TMM was used here. To determine whether a compensatory effect of IMCI/IControl (plots to the right of current traces) were calculated by aligning the current traces to the time of 1 mM glutamate application and calculating the point-by-point ratio (similar to80) of IMCI divided by IControl. IControl was calculated as the point-by-point average of IControl1 (current activated by 1 mM glutamate before MCI) and IControl2 (current activated by 1 mM glutamate after recovery from MCI).Data were analyzed with Clampfit 10.7 (Molecular Devices), Origin 16 or GraphPad Prism 7. Plots of IMCI/IControl was calculated as follows: (a) the minimum value of IMCI/IControl was located and the mean IMCI/IControl value over a 30 ms window centered on the minimum value was calculated; (b) the resulting value was normalized to the minimum value of control point-by-point ratios.The value of Min IMCI/IControl values were biased to be <1 (even without inhibition) because we selected the minimum IMCI/IControl value for measurement. In all experiments except pH jump experiments, the minimum value of control point-by-point ratios was calculated as the average of the minimum value (averaged over a 30 ms window) of the point-by-point ratios IControl2/IControl1 and IControl1/IControl2. We averaged minima of IControl2/IControl1 and IControl1/IControl2 to control for any possible rundown or runup of NMDAR response. For pH jump experiments, to control also for possible delayed effects of the pH jump on NMDAR responses, the minimum IMCI/IControl value was measured using an additional set of experiments. In the additional control experiments, an identical pH jump protocol was performed except with [memantine] = 0; the procedure described above for determining minimum value of point-by-point ratios then was used. The minimum value of IMCI/IControl in control (0 memantine) experiments used for normalization were: 0.906 ± 0.031 (n = 6) for the control pH 9.0 jump; 0.931 ± 0.035 (n = 5) for the control pH 6.3 jump.Normalization to the minimum value of control point-by-point ratios was performed because unnormalized minimum IControl2 and peak IControl1 differed by >20%. To minimize series resistance error, in addition to use of series resistance compensation cells were excluded from analysis if peak NMDAR current was >2.5 nA or if series resistance was >20 MΩ. Cells also were excluded from analysis if holding current was more negative than −200 pA  or if holding current fluctuations exceeded 100 pA during an experiment (to minimize inaccurate current quantification due to variation in holding current).To avoid inaccurate current quantification due to response run-down or run-up, MCI measurements were excluded if peak I50 values for WT receptors at pH 7.2 and 9.0  is the value of Min IMCI/IControl measured with [Mem]; A is the value of Min IMCI/IControl at saturating [Mem]; IC50 is the memantine MCI IC50; nH is the Hill coefficient. Equation  free. The value of A was constrained to 0; nH was constrained to 1.Memantine MCI IC9.0 Fig.  and for 9.0 Fig.  were cal9.0 Fig.  to [memaes Figs. g and 6g.col Fig.  allowed 6.3 Fig.  was calc50s  is fractional inhibition at [drug]; IC50 is the memantine, TMM, or DMM IC50; nH is the Hill coefficient. The equation was fit to Idrug/IControl values at all [drug]s from each cell. Free parameters during fitting were IC50 and nH.Traditional memantine IC50s Fig. , TMM IC5ting Eq.  to , 1.22 , 1.27 , 1.37-1.47 , 1.58 , 1.63 , 1.83 , 2.33 , 2.80 . 13C-NMR  δ: 30.2 , 31.5 , 34.0 , 35.8 , 37.2 , 42.7 , 43.1 , 50.6 , 66.1 . HRMS-ESI + m/z [M + H]+ calcd for [C14H26N]+: 208.206, found: 208.206. Anal. Calcd for C14H25N ∙ HCl·H2O: C, 64.22; H, 10.78; N, 5.35. Found: C, 64.29; H, 10.61; N, 5.18.A solution of memantine  in 15 ml of absolute EtOH was placed in a round-bottom flask equipped with a magnetic stirrer, a reflux condenser and an addition funnel. The solution was heated to 50 ˚C and formic acid  was added slowly for 30 min. After that, formaldehyde  was added dropwise with vigorous stirring for 1.5 h. When the addition of formaldehyde was completed, the reaction mixture was heated at 80 ˚C for 20 h. The resulting solution was then tempered to room temperature and the pH was adjusted to 12 with 5 M NaOH (10 ml). Dichloromethane (DCM) (30 ml) was then added, the phases were separated, and the aqueous phase was extracted with further DCM (2 × 10 ml). The combined organic phases were dried over anhydrous NaN,N,3,5-tetramethyladamantan-1-amine  in toluene (6 ml) at room temperature and stirred for 1 h. The white wax was filtered under vacuum and washed with toluene (10 ml) to yield a white solid . The analytical sample was obtained as a white solid by crystallization from methanol/diethyl ether and contained less than 1 ppm of memantine and less than 50 ppb of DMM as determined by HPLC/MS. Mp > 200 °C (dec.) (reported 290–293 °C)81. IR (ATR) ν: 3017, 2953, 2917, 2888, 2862, 2833, 1489, 1478, 1465, 1448, 1412, 1362, 1343, 1310, 1282, 1266, 1225, 1182, 1172, 1163, 1132, 991, 958, 947, 932, 920, 902, 842, 832, 799, 757. 1H-NMR  δ: 0.99 , 1.21–1.29 , 1.39 , 1.47 , 1.39 , 1.47 , 1.98 , 2.39 , 3.05 . 13C-NMR  δ: 30.3 , 32.2 , 34.3 , 34.8 , 41.6 , 42.3 , 49.05 (CH3), 49.09 (CH3) and 49.2 (CH3) [N(CH3)3], 50.2 , 75.7 . HRMS-ESI + m/z [M + H]+ calcd for [C15H28N]+: 222.2216, found: 222.2217. Anal. Calcd for C15H28IN: C, 51.58; H, 8.08; N, 4.01. Found: C, 51.58; H, 7.96; N, 3.89.Methyl iodide  was added dropwise to a solution of 50s of TMM and DMM are substantially higher than memantine’s, DMM and TMM were extensively purified to avoid artifactual NMDAR inhibition by memantine contamination of DMM or TMM, or by DMM contamination of TMM. Products were recrystallized from methanol/diethyl ether twice and the purity of the samples was followed by HPLC/MS with limits of detection (LOD) of 1 ppm for memantine and 50 ppb for DMM. After the recrystallizations, DMM contained less than 1 ppm of memantine while TMM contained less than 1 ppm of memantine and less than 50 ppb of DMM.Because the traditional ICSee Supplementary Methods and Supplementary Figs. 82. We used the FF12SB force field83 (for open state NMDAR Model 1) or the FF14SB force field84 (for open state NMDAR Model 2) for protein, the Lipid14 force field for lipids85, the TIP3P water model, and the GAFF force field parameters for memantine (developed using the Antechamber module of AMBER). Channel opening simulations for Model 1 were performed with a 1 fs integration step. For all other simulations an integration step of 2 fs was used and all covalent bonds to hydrogen atoms were constrained via SHAKE86. The Langevin thermostat and the Berendsen barostat87 with anisotropic pressure scaling were used to maintain temperature and pressure. Long range electrostatic interactions were calculated using the Particle Mesh Ewald method with a cutoff radius of 10 Å. Periodic boundary conditions were applied in all directions. All simulations were performed with initial minimization of the systems using the steepest descent algorithm, followed by MD at 1 atmosphere pressure and 300 K temperature in constant-temperature, constant-pressure (NPT) ensemble.All MD simulations were carried out using the pmemd.cuda program in the AMBER18 molecular dynamics package52. The model contained residues 559 to 657 and 809 to 838 of GluN1 and residues 554 to 655 and 813 to 842 of GluN2A. The full simulated system contained 522 protein residues, 108 DMPC membrane lipid molecules, and 10159 water molecules and Na+ and Cl− ions, resulting in a total of 43674 atoms. To obtain an open channel, a 10-ns steered MD simulation was performed (in 10 steps of ~1 ns each) with harmonic constraints applied to the SYTANLAAF sequence of all M3 helixes. The constraints were designed to gradually increase the distance between M3 helixes at the channel gate until the channel filled with water. To maintain structural integrity of the protein, backbone hydrogen bonds and dihedral angles of all TMD helices were harmonically restrained with a force constant of 20 kcal mol-1Å-2. The protocol was similar to the one used to produce an open AMPAR model53.To develop open state NMDAR Model 1, a MD-optimized closed channel model of the GluN1/2A NMDAR TMD in lipid bilayer and water was taken from our previous work54 (PDB ID: 6MM9 [https://www.rcsb.org/structure/6mm9]) was used as the starting template. The SYTANLAAF sequence of M3 helices of Model 2 were modeled based on the cryo-EM structure of an open AMPAR55 (PDB ID: 5WEO [https://www.rcsb.org/structure/5WEO]). The open AMPAR structure contains an asymmetric external gate with kinked M3 helices in the B and D subunits, which correspond to the GluN2A subunits of our NMDAR model. Model 2 contained residues 550 to 657 and 809 to 838 of GluN1 and residues 545 to 655 and 813 to 837 of GluN2A. The model was placed in an equilibrated POPC lipid bilayer using CHARMM-GUI 3.2 Membrane Builder89. The full system in lipid bilayer and water contained 548 protein residues, 426 POPC membrane lipid molecules, 34413 water molecules and Na+ and Cl− ions, resulting in a total of 169075 atoms. The open channel was equilibrated with gradually decreasing harmonic restraints on the protein backbone from 20 to 0.05 kcal mol-1 Å-2 over 100 ns followed by unrestrained MD simulations for 400 ns.To develop open state NMDAR Model 2, the cryo-EM structure of a closed state GluN1/2A NMDAR56 was used to identify possible memantine paths from membrane to channel in representative structures of both open state NMDAR models. Multiple snapshots extracted from the equilibrium MD trajectory of Model 2 were analyzed with HOLE to select a structure with optimal side chain orientations for docking and subsequent MD simulations. Memantine (uncharged) was docked to the open state NMDAR models with AutoDock Vina 1.2.091 at 5 Å intervals along the path identified by HOLE using a grid box of 16 Å × 16 Å × 16 Å centered around path-lining residues. Each docked complex was energy-minimized and equilibrated for 10 ns with all protein Cα atom positions restrained with a force constant k = 1.0 kcal mol−1 Å−2. Steered MD simulations were carried out in two stages: (1) Memantine was pulled from the docked site at the path entrance into the protein by gradually decreasing the distance between the center of mass (COM) of memantine and Cα atoms of protein residues GluN2A(A604) and GluN2A(V631) from ~15 Å  to 0 Å. (2) Memantine was pulled from the docked site near GluN2A(M630) into the channel by gradually decreasing the distance between the COM of memantine and the COM of protein residues GluN1(M641) and GluN2A(V639) from ~20 Å until the ligand entered the ion channel. The Cα atom positions of helical segments of the protein were restrained using a force constant k  =  40.0 kcal mol−1 Å−2 during both stages. The trajectories from the above two stages were combined to obtain the full path of memantine from the membrane to the channel. Two separate sets of “pulling simulations” were carried out with the following parameter values: (1) k = 10 kcal mol−1 Å−2 (k = 5 in AMBER) and t = 60 ns, and (2) k = 4 kcal mol−1 Å−2 (k = 2 in AMBER), t = 100 ns, where k = the biasing force constant and t = the total simulation duration. VMD 1.9.492 was used to visualize trajectories and generate molecular graphics.The program HOLE v2.2post hoc analysis and two-tailed t-tests as indicated. For all electrophysiological experiments, n is the number of biologically independent cells. All error bars indicate ± standard error of the mean (SEM). Mean and SEM values for MCI IC50s (Figs. MCI/IControl mean ± SEM values (Figs. Statistical tests were performed in GraphPad Prism 7. The same sample was not measured repeatedly. We used one-way ANOVA with Tukey’s 0s Figs. h and 6h es Figs. g and 6g.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplmentary FilesSupplementary Movie 1Reporting Summary


Does representative hazardous-waste-site testing tend to follow or to violate government technical guidance? This is an important question, because following such guidance promotes reliable risk analysis, adequate remediation, and environmental-justice and -health protection. Yet only government documents typically address this question, usually only when it is too late, when citizens have already exhibited health harm, allegedly from living or working near current/former hazardous-waste sites. Because no systematic, representative, scientific analyses have answered the preceding question, this article begins to investigate it by posing a narrower part of the question: Does representative US testing of volatile-organic-compound (VOC) waste sites tend to follow or to violate government technical requirements? The article (i) outlines US/state-government technical guidance for VOC testing; (ii) develops criteria for discovering representative US cases of VOC testing; (iii) uses the dominant US Environmental Protection Agency method to assess whether these representative cases follow such guidance; (iv) employs the results of (iii) to begin to answer the preceding question; then (v) discusses the degree to which, if any, these results suggest threats to environmental health or justice. Our initial, but representative, results show that almost all US VOC-waste-site testing (that we investigated) violates government technical requirements and systematically underestimates risks, and this may help justify less expensive, potentially health-threatening cleanups, mostly in environmental justice communities. We outline needed future research and suggest two strategies to promote following government technical guidance for hazardous-waste testing. Early in 2018, the 40,000-member African-American community living in homes built on San Francisco’s former Hunter’s Point hazardous-waste site filed a $27 billion class-action lawsuit against the contractor Tetra Tech. The suit accused the corporation of falsifying sampling and, as a result, causing community members’ environmental injustice, cancer, and death .First, although many governments monitor hazardous-waste generators, the reliability of site testing/cleanup is mainly unknown; scientific databases reveal no systematic, representative, third-party studies that empirically assess hazardous-waste testing. Second, as Third, because of this privatization, government typically does not independently audit hazardous-waste sampling  and the main potential environmental-health and environmental-justice consequences, if any, of following, versus violating, VOC-testing guidance.The question about the prevalence of flawed waste-site testing arose for the author in spring 2018, when members of the mostly poor minority community living near a hazardous-waste site—the former Naval Ordnance Testing Station, Pasadena, California, NOTSPA see  below—reBecause NOTSPA is both a VOC hazardous-waste site and is surrounded by a typical environmental justice community see  below, iThe NOTSPA developer’s testing documents reveal that chlorinated solvents—particularly the VOCs trichloroethylene (TCE), perchloroethylene (PCE), carbon tetrachloride (CT), and dibromochloromethane (DBCM), the four highest site risks—are ubiquitous in site soil, at concentrations up to nearly one million times above allowed levels. For decades, state regulators have warned of NOTSPA vapor intrusion (VI), migration of subsurface VOC carcinogens/neurotoxins into above-ground buildings. As a result, regulators filed an Imminent and Substantial Endangerment Order because of VI threats to site renters and nearby residents. However, the site has never been cleaned up see .Like NOTSPA, hazardous-waste sites (and whether their testing/cleanup follows regulatory guidance) affect environmental-justice (EJ) communities because most toxic-waste sites throughout the world are surrounded by EJ communities. Thus, if pollution is not properly tested/remediated, EJ communities bear the highest risks. That is, statistically, the closer one lives to a hazardous-waste site , the more likely that one is a member of an EJ community. EJ communities are those whose members, as compared to the rest of the local population, tend to bear higher levels of pollutants, have poorer health, be members of minority groups, have lower socioeconomic status, be unemployed, have lower levels of education, or have several of the preceding characteristics .The California state government’s CalEnviroScreen data show that the census tract (in which NOTSPA is located) is a typical EJ community. Regarding unemployment, in pre-COVID 2019, the average California unemployment rate was 4%, but the NOTSPA census-tract unemployment rate was 600% higher . RegardiUnsurprisingly, some of the health risks in the NOTSPA census tract may be at least partly attributable to NOTSPA-site pollutants. For instance, the disproportionately high incidence of low-birthweight infants in this census tract (350% higher than the state average ) may ariBesides illustrating the importance of reliable waste-site testing and its potential environmental health/justice characteristics, NOTSPA also illustrates why most US hazardous sites have not been remediated. Although NOTSPA must follow US CERCLA-cleanup requirements, like most waste sites, it has not been remediated. Of 1714 US CERCLA/superfund sites, only 387 (23%) have been remediated ,12,13. OOne reason is that some decisionmakers have decided that immediate hazardous-site reuse has economic benefits that “outweigh…significant and unavoidable” hazardous impacts; as a result, roughly 9000 brownfields are “ready for reuse,” but not remediated ,16,17  analyses to begin systematically investigating hazardous-site VOC testing before potential health harm appears. Our study objective is to provide a preliminary answer to the question: Does representative testing at US subsurface VOC-contaminated sites tend to follow or to violate the “requirements” of the US regulatory technical guidance for VOCs? .e, e.g., .For critical environmental-health reasons, our question deserves analysis. A full 23% of US citizens, 54% of minorities, and 25% of children under age 5, live within 1 mile of brownfields or US CERCLA sites . Yet as Health and EJ threats from VI continue partly because the financial incentives in the 2002 US Brownfields Revitalization Act may have created a conflict of interest : Privatemethod can we use to evaluate our question, “Does representative US VOC-waste testing meet regulatory-testing requirements?” Because answering this question is not amenable to randomized, controlled trials—and because we must weigh multiple pieces of pre-existing evidence through meta-analysis, not merely generate evidence—we use the hazardous-waste assessment method developed/recommended by the US Environmental Protection Agency (EPA). This is the systematic, 2016, three-part, weight-of-evidence (WoE) method. Employed by most US hazardous-site assessors, as well as risk and environmental-impact assessors, WoE is EPA’s main method for weighing multiple pieces of often-conflicting lab, field, statistical, or modeling evidence to infer policy-relevant or potential regulatory actions  data for evaluating vapor intrusion should meet the following [six] requirements” or compulsory actions. These are that all sampling mustsources”  dvisory” ,46,including soil-gas testing that:contaminants are no longerencountered,” thus providing “subsurface,” “three-dimensional” toxin delineations;(R4.1) continues “until vapor-phase two-sub-slab sampling events” at all building centers;(R4.2) provides a “minimum, maximum subsurface concentrations” [(R4.3) locates all “rations” .reporting limits “lower than health-protective [screening] levels”  isoconcentration contours” ,46.The WoE Method Part 3 uses its Part-2 results  to assess testing at five VOC-waste sites. Recall that the five VOC-waste testing sites, discovered through WoE Part 1, are located in Pasadena (NOTSPA), Monrovia, Canoga Park, Pacoima, and Boyle Heights, California. Thus, total possible testing violations number 30 for all five hazardous-waste sites.Subsequent paragraphs show that, surprisingly, both Pasadena (NOTSPA) and Monrovia hazardous-waste-site testing violate all six of the preceding six regulatory requirements for VOC testing (R1–R6). The main contaminants at both sites are VOCs  (p. 34);Though NOTSPA “risk drivers” are chlorinated-VOC solvents and carcinogens CT, DBCM, PCE, and TCE  (p. 34),−6 risk level, the health-protective, residential-screening level required for the planned 550 NOTSPA apartments; yet 40% of these apartments are for families with children  mitigation [not removal] will be performed, as necessary” (author’s bold) …DTSC is not properly enforcing state and federal law…. Numerous statutory changes have been made to…address outstanding programmatic failings. However, many of the underlying concerns about [DTSC] transparency… [and] accountability…remain” (pp. 6–7Another example of DTSC-oversight failures is Exide Battery in East Los Angeles (LA), one of poorest, most-polluted Latino communities, and home to disproportionate numbers of children. For 30 years and without requiring any testing, DTSC claimed Exide’s impacts on East LA were “less than significant.” Only in 2020 did nearby victims force DTSC to issue an Imminent and Substantial Endangerment Order, to recognize Exide’s four felonies, its illegal releases of 3500 tons of lead, and its causing permanent IQ deficits and chronic health harm among 250,000 nearby residents, mostly children. Because of failed DTSC oversight, thousands of East LA children now have unsafe blood-lead levels (double the LA average lead level) that will limit their opportunities in life ,94.3 TCFM SL . −6 cleanup level ,TC claims NOTSPA “soil outside the storm-drain system is expected to be non-hazardous”  (p. 42),TC claims NOTSPA northern soil is “clean”  (p. 60),TC officially claimed and wrote to Pasadena City Council that “all of the [toxin-] impacted soils” will “be removed” from the NOTSPA site , yet itsAt NOTSPA, TC likewise claimed it “extensively investigated and tested” and “appropriately tested…the entire site,” although it violated all six of six DTSC soil-gas VOC testing requirements see . TC alsoIn summary, TC’s misrepresentations of its hazardous-site testing and DTSC’s regulatory-oversight deficiencies may exacerbate TC’s apparent violations of VOC-testing requirements. As a result, they may pose additional threats to environmental health.the California regulator’s CalEnviroScreen, the online multiple-pollutant indicator, ranks Pacoima and Boyle Heights toxic site censustracts among California’s 4% most polluted  results” . If CaliDoes US hazardous-waste testing (most of which is privatized), at representative US subsurface-VOC sites, tend to follow or to violate regulatory “requirements” for VOC testing?Our results (29 of 30) reveal a representative, conservative sample of privatized-testing violations that answer our study question in the affirmative: If our results can be replicated, they suggest that inadequate waste-site testing may put at risk both environmental health and EJ. Flawed waste testing/cleanup may force some members of EJ communities to choose between hazardous housing and no housing. Our analysis also suggests that privatized cleanups may face difficulties like those of private prisons. California’s governor recently abolished private prisons, claiming they are “driven to maximize…profits… [and] lack proper oversight” . Does pr


Radiolucent carbon-fiber reinforced PEEK (CFRP) implants have helped improve oncological follow-up and radiation therapy. Here, we investigated the performance of 3D intraoperative imaging and navigation systems for instrumentation and precision assessment of CFRP pedicle screws across the thoraco-lumbar spine. Thirty-three patients with spinal tumors underwent navigated CFRP instrumentation with intraoperative CT (iCT), robotic cone-beam CT (rCBCT) or cone-beam CT (CBCT) imaging. Two different navigation systems were used for iCT-/rCBCT- and CBCT-based navigation. Demographic, clinical and outcome data was assessed. Four blinded observers rated image quality, assessability and accuracy of CFRP pedicle screws. Inter-observer reliability was determined with Fleiss` Kappa analysis. Between 2018 and 2021, 243 CFRP screws were implanted , of which 13 were non-assessable . Navigation accuracy was highest using iCT (74%), followed by rCBCT (69%) and CBCT (49%) . All observers rated iCT image quality higher than rCBCT/CBCT image quality (*p < 0.01) but relevant pedicle breaches were reliably identified with substantial agreement between all observers regardless of the imaging modality. Navigation accuracy for CFRP pedicle screws was considerably lower than expected from reports on titanium implants and CT may be best for reliable assessment of CFRP materials. Against this background, technology in the field of spine surgery has evolved towards the development of new implant materials and computer-assisted image guidance: Carbon fiber reinforced PEEK (CFRP) has been reported to have biomechanical screw properties comparable to standard titanium implants8 with distinct advantages of reduced imaging artifacts9, less perturbation effects10 and the potential to significantly improve planning, safety and quality of adjuvant radiotherapy and follow-up16. Real-time spinal navigation with intraoperative 3D imaging has improved pedicle screw accuracy22, reduced radiation exposure for the OR team23, and holds promise to improve outcomes24. Importantly, the utilization of state-of-the-art intraoperative 3D imaging also permits immediate implant control and direct revision, if needed. Three of the most widely used intraoperative imaging solutions are the mobile AIRO intraoperative CT (iCT)25, the permanently installed Zeego robotic cone-beam CT (rCBCT)26, and the mobile O-arm cone-beam CT (CBCT)28. So far, little is known about the performance of using intraoperative 3D imaging in the context of CFRP pedicle screw implantation. This is highly relevant, however, because performance may differ compared to titanium screws due to the radiolucency and specific surgical nuances are required for CFRP screw insertion29. Therefore, in the present study we describe the performance of iCT, rCBCT- and CBCT-based spinal navigation for CFRP screw implantation in patients suffering oncologic spinal disease.For patients with spinal oncological disease, posterior pedicle screw fixation with or without circumferential decompression, en-bloc spondylectomy, or anterior cage reconstruction represents an accepted strategy to address tumor burden, cord compression, instability and pain30, as well as surgical data, navigation accuracy and screw assessability for each imaging modality were retrospectively analyzed. Informed consent was waived by the ethics committee of the Charité—Universitätsmedizin Berlin according to EA4/046/16 and EA4/063/20 due to the retrospective nature of the study.All methods were performed in accordance with the relevant guidelines and regulations. This single-center retrospective cohort study was conducted according to the World Medical Association Declaration of Helsinki in compliance with Health Insurance Portability and Accountability Act regulations and approved by the local ethics committee of the Charité—Universitätsmedizin Berlin (EA4/046/16 and EA4/063/20). Between January 2018 and March 2021, 33 patients with oncological spinal pathologies underwent implantation of 243 CFRP pedicle screws using iCT-, rCBCT- or CBCT-based real-time spinal navigation. The inclusion criteria for this retrospective analysis were spinal metastases or primary spinal tumors that were treated with navigated instrumentation using CFRP pedicle screw implants with or without combined corpectomy at the level of the thoracic or lumbar spine. The choice of intraoperative imaging modality was based on logistical considerations and availability. Importantly, specific anatomical or pathological considerations like the anatomic region or type of tumor did not affect the choice of imaging. The decision to use CFRP implants was based on availability and the presence of oncological spinal disease requiring posterior pedicle screw fixation with high likelihood of requiring continuous oncological imaging follow-up and/or adjuvant irradiation therapy planning. Clinical, demographic and tumor data according to the NOMS framework32. For CBCT-based spinal navigation, the mobile O-arm system  was used. For navigated pedicle screw implantation, an image-guidance system and infrared tracking camera with automatic patient/image co-registration was used 28.For iCT- and rCBCT-based spinal navigation, the AIRO iCT  and robotic 3D Artis Zeego II digital fluoroscopy C-arm system  were used as previously described28.Surgery was performed on mobile, radiolucent, carbon-fiber examination tables . For all rCBCT procedures, the patients’ head was fixed in a radiolucent carbon fiber 3-pin head clamp . Surgical exposure was gained through a standard midline approach and a navigation reference clamp  was attached to a spinous process. The screw entry point and trajectory were identified with a navigated drill-guide . A battery-powered drill  with a 2.6 mm drill bit was used to drill a pilot hole down to a desired depth. Next, a guide-wire was inserted, the pedicle was tapped, and a cannulated, CRFP pedicle screw with a diameter between 5.5 and 7.5 mm  was inserted Fig. . Screw pExpert observer, according to the 2 mm-increment method initially described by Gertzbein and Robbins33 and modified according to Rampersaud34. Categories A (completely within the pedicle) and B (< 2 mm pedicle breach) were classified as accurate and categories C (2–4 mm pedicle breach) and D (> 4 mm pedicle breach) as inaccurate placement.Definite screw accuracy rates and information on general screw assessability were based on the assessment obtained from the most experienced For the analysis of inter-observer reliability, anonymized, intraoperative imaging data sets of all patients were generated from the hospital PACS system. In addition to anonymization, patient data was blinded towards the used intraoperative imaging modality . The anonymized and blinded image data sets were then distributed to four independent observers: two observers on expert-level , one senior PGY6 resident , and one PGY1 junior resident . Image assessment was performed with a dedicated DICOM viewer . For every observer and each patient, the perceived image quality , general screw assessability (yes/no), perceived pedicle screw accuracy (grading according to modified Rampersaud A-D), as well as the time required for accuracy assessment beginning from the 3D image reconstruction until the completion of the assessment (minutes) were determined. Screw accuracy and assessability analysis of each observer was compared for its consistency with calculation of Fleiss’ Kappa inter-observer agreement.For data management and blinded analysis, REDCap  was used. Anonymized and blinded DICOM datasets were stored on encrypted portable devices. For statistical analysis, SPSS Version 25  and GraphPad Prism Version 9  were used. All tests were two-sided. Statistical significance was set at p < 0.05 and tested by one-way ANOVA with Bonferroni correction for multiple comparisons or Kruskal–Wallis test with Dunn’s correction for multiple comparisons, depending on normal distribution according to the Shapiro–Wilk test. Inter-observer reliability was tested with Fleiss’ Kappa analysis, with interpretation of reliability by Landis & Koch .The study was approved by the ethics committee of the Charité-Universitätsmedizin Berlin, Germany (EA4/046/16 and EA4/063/20). Waived due to the retrospective nature of the study.Detailed demographic, clinical, tumor and outcome data are displayed in Table The number of generally assessable screws differed significantly between iCT (99%), rCBCT (91%) and CBCT . Six out of 93 (7%) iCT screws underwent navigated revision, in contrast to zero revised screws in the rCBCT and CBCT groups (Table Experts and Residents) and found that both Experts and Residents rated the image quality of iCT higher compared to CBCT technology  but this did not relevantly affect the time required for screw accuracy analysis  permitted highly sensitive and specific identification and categorization of even the slightest pedicle breaches. In particular, radiolucent CFRP implants offered a completely distinct and sharp view of the pedicle isthmus outline and permitted highly precise measurement and breach categorization according to the perception of the most experienced Expert observer, who graded the baseline categorization. Since the majority of all misplacements were noted within the range of 2–4 mm (category C), such highly sensitive breach detection and categorization might have unmasked category B (or C) CFRP screws that would otherwise have been graded as category A (or B) using titanium implants. Second, implantation of CFRP pedicle screws requires knowledge of certain technical nuances for successful implantation, such as meticulous selection of the screw entry point in order to ensure precise alignment, given the inability to bend the pre-shaped carbon fiber rods that we used16. Third, in oncologic spinal disease we typically aim to implant the largest possible screw diameter just below the pedicle isthmus diameter to ensure maximum pullout resistance and limit the risk of a pedicle breach. In the present cohort, the median pedicle isthmus diameter of breached pedicles ranged between 5.7 and 6.1 mm for all imaging modalities  to 74% (iCT) that we experienced with CFRP pedicle screws was unexpected, considering that previous studies have consistently demonstrated navigation accuracy rates above 90% for titanium implants using each of the three investigated imaging modalities38 and we believe that continuous exposure is required to maintain a high level of performance and ensure training of the entire surgical team, including spine surgeons and residents with different levels of experience. The radiolucency of CFRP implant materials present a challenge regarding simple, fast and reliable assessment of pedicle breaches. Therefore, we determined the inter-observer reliability between resident and expert observers as well as the perceived image quality and required time for accuracy assessment of CFRP pedicle breaches based on iCT, rCBCT and CBCT imaging. The finding that residents generally rated screw placement accuracy with less agreement than experts and that agreement was lowest for CBCT imaging seems intuitive, given the likely greater experience of fully trained spine surgeons in judging pedicle screw accuracy compared to residents in training and the generally lower perceived image quality of CBCT compared to iCT. On the other hand, the fact that residents had substantial to almost perfect agreement when grading breaches considered most likely to be clinically relevant (categories C and D) and that this high level of agreement was reached for each imaging modality, including CBCT, nicely shows that each of the imaging modalities is feasible for reliable detection of clinically relevant breaches, regardless of the observers’ level of experience and despite differences in image quality.Inadequate training is one of the main reasons cited when spine surgeons refrain from adapting image-guidance2]), which prohibits a direct comparison of system-documented radiation dosage.The retrospective design of our study and small sample size bears well known limitations and lacks systematic outcome assessment, including pain and Quality of Life scores and follow-up regarding implant durability. Another major limitation is that the individual effective radiation (organ) doses applied by each modality were not directly measured, so that individual radiation exposure was not comparable, because radiation dosage in fan-beam CT (iCT) and cone-beam CT (rCBCT/CBCT) are recorded in different dosage units (iCT: dose-length-product [mGy/cm]; rCBCT/CBCT: dose-area-product [mGycmAccuracy of navigated CFRP pedicle screws was considerably lower than expected from previous navigation experience with titanium implants and CFRP screw assessability as well as the interrater-reliability of CFRP screw assessment were affected by the type of imaging modality and the experience of the observer. Overall, iCT yielded the highest navigation precision, best perceived image quality and permitted robust CFRP implant assessment independent from the users’ experience. The present study shows that the choice of intraoperative 3D imaging for navigated CFRP pedicle screw instrumentation has significant impact on standard procedures in the field of computer assisted spine surgery.


The aim of the study is to develop a nomogram for predicting postoperative portal venous systemic thrombosis (PVST) in patients with cirrhosis undergoing splenectomy and esophagogastric devascularization.  In total, 195 eligible patients were included. Demographic characteristics were collected, and the results of perioperative routine laboratory investigations and ultrasound examinations were also recorded. Blood cell morphological traits, including the red cell volume distribution width (RDW), mean platelet volume, and platelet distribution width, were identified. Univariate and multivariate logistic regressions were implemented for risk factor filtration, and an integrated nomogram was generated and then validated using the bootstrap method.  A color Doppler abdominal ultrasound examination on a postoperative day (POD) 7 (38.97%) revealed that 76 patients had PVST. The results of the multivariate logistic regression suggested that a higher RDW on POD3 (RDW3) (odds ratio (OR): 1.188, 95% confidence interval (CI): 1.073–1.326), wider portal vein diameter , history of variceal hemorrhage , and longer spleen length  were independent risk parameters for postoperative PVST. Moreover, the nomogram integrating these four parameters exhibited considerable capability in PVST forecasting. The nomogram's receiver operating characteristic curve reached 0.83 and achieved a sensitivity and specificity of 0.711 and 0.848, respectively, at its cutoff. The nomogram's calibration curve demonstrated that it was well calibrated.  The nomogram exhibited excellent performance in PVST prediction and might assist surgeons in identifying vulnerable patients and administering timely prophylaxis. Howeverery year . Splenecery year . For patery year . Thus, SHowever, portal venous systemic thrombosis (PVST)  is a common and serious complication following SED, with an incidence rate ranging from 18.9% to 55% , 7. FurtTo date, studies investigating PVST following SED have reported various risk factors, including a wider portal vein diameter (PVD), poorer liver function, prolonged prothrombin time (PT), higher levels of preoperative aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and postoperative thrombocytosis –14. HoweThis single-center retrospective case-control study conformed to the Declaration of Helsinki (as revised in 2013) and was approved by the Medical Ethics Committees of the Second Hospital of Nanjing.9/L, white blood cell (WBC) count <3 × 109/L, and/or with a history of variceal bleeding resulting from PH, and (5) patients with a Child-Pugh score of A or B.Patients with cirrhosis who underwent open SED at the Department of Hepatobiliary Surgery, Second Hospital of Nanjing, between January 2013 and December 2018 were included. A detailed description of the SED procedure is provided in the following section. The inclusion criteria were as follows: (1) patients aged between 18 and 75 years, (2) patients diagnosed with liver cirrhosis based on pathological or radiological evidence, (3) patients with PH and severe EV , (4) patients with a platelet count <50 × 10The exclusion criteria were as follows: (1) patients with hepatocellular carcinoma or other malignant diseases identified before or during surgery, (2) patients with PVST prior to surgery, (3) patients with hematological or immune system diseases, (4) patients who had received anticoagulants prior to surgery, (5) patients with organ failure or refractory ascites, and (6) patients with missing data.Basic data including age, sex, body mass index (BMI), history of hypertension, history of diabetes mellitus, history of variceal hemorrhage, cirrhosis etiology, the presence of ascites, emergency at admission, the model for end-stage liver disease (MELD) score, Child-Pugh score, PVD, spleen thickness, spleen length, and portal venous flow velocity were recorded. Preoperative laboratory blood tests to measure WBC, red blood cells (RBCs), RDW, platelet distribution width (PDW), mean platelet volume (MPV), hematocrit, coagulation parameters, AST, ALT, total bilirubin, albumin, globin, and postoperative RDW, PDW, MPV, and hematocrit were also collected on a postoperative day (POD) 1, 3, and 7.All the patients received an abdominal color Doppler ultrasound examination on POD7, and the diagnosis of PVST was confirmed by two experienced imaging experts. Ultrasound follow-up was conducted every 2 weeks for all patients.All patients underwent SED using laparotomy. The abdomen was opened using an L-shaped left subcostal incision, and a splenectomy was performed. The peripheral ligaments of the spleen, including the gastrocolic ligament, splenocolic ligament, gastrosplenic ligament, splenorenal ligament, and splenophrenic ligament, were cut and suture ligated. The splenic hilum was rigorously dissected, and the splenic artery and vein were carefully transected and ligated. Soon after splenectomy, devascularization was conducted. The portosystemic collateral branches at the lower esophagus and fundus of the stomach, including the short gastric vein, posterior gastric vein, left inferior phrenic vein, and esophagogastric branches of the gastric coronary vein, were identified and ligated. The esophagus was dragged down, and the high esophageal branches of the left gastric vein were sutured at a distance of approximately 10 cm from the fundus of the stomach; the arteries accompanying these veins were divided accordingly.t-test was performed on data with normal distribution, and the Mann–Whitney U test was used for nonparametric tests. Categorical variables were compared using either the Chi-square test or Fisher's exact test. Normally distributed continuous variables are presented as the mean (standard deviation) and nonnormal variables as the median . Categorical variables are presented as the exact number . Significant variables in the univariate comparison were further analyzed using a multivariable stepwise logistic regression.The normality of continuous data were analyzed using the Shapiro–Wilk test. The Student's p value of less than 0.05 was considered significant throughout the analysis. R software (version 3.6.1) was used for all the statistical analyses.The nomogram was developed based on the multivariate logistic regression model using the “rms” R package. The bootstrap method   of the patients. The commonest cause of cirrhosis was infection with hepatitis B (140 patients), with the other causes being hepatitis C infection (34 patients), alcoholic hepatitis (14 patients), primary biliary cirrhosis (4 patients), drug-induced cirrhosis (2 patients), and schistosomiasis cirrhosis (1 patient).All patients underwent SED using laparotomy . Detailed data from the PVST and non-PVST subgroups are presented in p=0.008). In the preoperative ultrasound imaging, the spleen length was greater in the PVST group than in the non-PVST group , and the PVD was wider in the PVST group . The PVST group had significantly lower hemoglobin levels at admission . RDW was significantly higher in the PVST group either before surgery  or on POD1 , POD3 , or POD7 . In comparison with the non-PVST group, the PVST group had lower values in the following parameters: lymphocyte count , hematocrit , fibrinogen , and AST .No significant differences were identified in the velocity of portal blood flow, spleen thickness, emergency operation rate, Child-Pugh score, MELD score, or ascites between the PVST and non-PVST groups. Regarding the preoperative laboratory indicators, no significant differences were detected in RBC count, neutrophil count, monocyte count, platelet count, MPV, PDW, PT, activated partial thromboplastin time, thrombin time, total bilirubin, ALT, albumin, or globin between the two groups. However, variceal bleeding was more common in the PVST group  of the nonsignificant variables, PVD , spleen length , bleeding history , and RDW on POD3 (RDW3)  were identified as independent risk factors for PVST. The results of the multivariate regression are displayed in A ROC analysis was conducted to validate the performance of the independent risk factors and logistic model in forecasting postoperative PVST. The ROC curves of the individual risk parameters are presented in The nomogram was generated using a logistic model . Each riPH and hypersplenism are common complications of cirrhosis. Splenectomy greatly reduces pressure in the portal system and ameliorates variceal bleeding and ascites in patients with cirrhosis; however, it also increases patients' exposure to infection –20. SpleOne study reported that patients undergoing SED had a relatively high incidence of postoperative PVST (18.9%–55% vs. 5%–15%) . The incTo date, multiple factors, such as postoperative thrombocytosis, a wider preoperative splenic vein diameter, faster portal blood flow, prolonged PT, larger spleen volume, and devascularization, have been reported as risk factors for postoperative PVST , 24 but This study identified that preoperative PVD was an independent risk factor for postoperative PVST . Previous studies , 25 havep < 0.001). Variceal hemorrhage is the manifestation of a decompensated liver, and patients with a history of bleeding have a relatively poor prognosis [p=0.006) but they failed to include this factor in their final model.This study also revealed spleen length as a risk factor for PVST . Spleen volume reflects the severity of PH and the degree of hypersplenism; the larger the spleen is, the more serious the PH, and thus, the greater the likelihood of PVST . In addirognosis . Previourognosis  have proThe RDW is one of the parameters in routine blood tests—it is an indicator of the variability in erythrocyte size and is frequently overlooked in thrombosis events. Recently, the role of the RDW in thrombotic diseases has attracted significant attention from researchers. Relevant studies have demonstrated that the RDW is a powerful indicator of myocardial infarction, cerebral thrombosis, pulmonary embolism, and atrial fibrillation –33. LappIncreasing evidence suggests that the RDW is a feasible surrogate indicator for systemic inflammatory response and oxidative stress –38. Relep=0.001). This may be because systemic inflammation is at its most severe on POD3 and is indicative of a high likelihood of PVST. An excessive inflammatory response may lead to severe epithelial damage, facilitating the adhesion of platelets. Furthermore, considering the higher postoperative RBC levels in the PVST group, it is hypothesized that the elevated RDW reflects the hemopoiesis attributed to the cytokines secreted during inflammation, and this may subsequently result in an increase in blood viscosity.This study identified that patients with postoperative PVST exhibited a higher RDW either before or after SED; however, only the RDW on POD3 was an independent risk factor for postoperative PVST (OR: 1.188; 95% CI: 1.073–1.326; The current consensus on the prophylaxis of postoperative PVST is to administer anticoagulants once the risk of bleeding is eliminated. Following splenectomy, the prophylactic use of anticoagulants, such as low-molecular-weight heparin and rivaroxaban, can help to reduce the occurrence of PVST . In our This study has some unavoidable limitations. First, because the study had a retrospective design and was conducted in a single center with a small sample size of only 195 patients, a selection bias may be inevitable. Second, the validation of the logistics model was based solely on interval bootstrapping methods. External large-scale sample validation and future multicenter prospective studies are warranted to test the broader application of the proposed model. Third, most of the candidate factors included in the multivariable analysis were selected from the literature, and the independent risk factors may be affected by the inclusion of additional candidates. Last, the value of the RDW is influenced by multiple factors not considered in this study, such as the absorbance of vitamin B12 and iron.In summary, we proposed a nomogram integrating preoperative PVD, a history of variceal hemorrhage, spleen length, and the RDW on POD3, which exhibited an excellent capability for identifying patients vulnerable to PVST soon after SED. Our nomogram may aid surgeons in forecasting postoperative PVST and administering timely prophylactic treatment to patients at high risk of thrombosis. In the future, appropriate large-scale external validation may confirm the utility of the proposed nomogram in PVST forecasting.


We report a high-resolution record of dust deposition and ocean productivity for the Antarctic Zone, close to the main dust source, Patagonia. Our deep-ocean records cover the last 1.5 Ma, thus doubling that from Antarctic ice-cores. We find a 5 to 15-fold increase in dust deposition during glacials and a 2 to 5-fold increase in biogenic silica deposition, reflecting higher ocean productivity during interglacials. This antiphasing persisted throughout the last 25 glacial cycles. Dust deposition became more pronounced across the Mid-Pleistocene Transition (MPT) in the Southern Hemisphere, with an abrupt shift suggesting more severe glaciations since ~0.9 Ma. Productivity was intermediate pre-MPT, lowest during the MPT and highest since 0.4 Ma. Generally, glacials experienced extended sea-ice cover, reduced bottom-water export and Weddell Gyre dynamics, which helped lower atmospheric CO A new high-resolution record for the Antarctic Zone shows persistent anti-phasing of high interglacial ocean productivity and high glacial dust deposition, suggesting a close inter-hemispheric coupling of cryosphere, ocean and atmosphere. Although it is commonly accepted that dust deposition increased in both hemispheres during glacials, the fertilizing effect remains controversial and region specific4. For the sub-Antarctic zone (SAZ) of the Southern Ocean there is evidence for enhanced ocean productivity during glacials7, consistent with the Fe fertilization effect. In contrast, for the Antarctic zone (AZ)—the productive but Fe-limited Southern Ocean south of the Antarctic Polar Front  fertilization from dust deposition2 to the surface ocean4. This could be the reason for enhanced interglacial productivity but due to Fe and light limitation, an inefficient biological pump might cause incomplete consumption of nutrients13. A more southerly position of Southern Hemisphere Westerly winds (SHW) over the core of the Antarctic Circumpolar Current (ACC) during interglacials, would support such enhanced upwelling14, and consequently lead to enhanced escape of CO2 to the atmosphere. Lower glacial productivity within the AZ could hence have been associated with more efficient nutrient consumption according to 15N measurements15 and a more northerly position of the SHW.High upwelling rates are observed for the modern AZ, supplying nutrients and deeply sequestered CO16 was aimed at improving our understanding of how the coupled Antarctic ice-ocean-atmosphere system evolved to the present state in the high latitude sea-ice region of the AZ  Expedition 382 “Iceberg Alley” AZ Fig. .Fig. 2Ag17, for which South America (Patagonia), Southern Africa and Australia are the major sources18 , radiocarbon dating and MS synchronization24, showed that age models from records in the Scotia Sea are “mutually consistent over their common ranges” on orbital and millennial time scales, and that fluctuations in marine MS and Antarctic dust concentration were synchronous. Studies on Sites MD07-3133 and MD07-3134 confirmed very close resemblance of the MS signal to the non-sea salt (nss) Ca2+ signal of the EDML ice core, a confident proxy for detrital dust in Antarctic ice cores25, and established the use of MS as high-resolution chronological control over the last glacial cycle20.Earlier studies have identified a remarkable similarity in the structure of variability of magnetic susceptibility (MS) in cores from the Scotia Sea and the variability in dust records from Antarctic ice cores20 by first synchronizing the marine dust proxy MS to the ice-core dust flux of the EDC ice core19 on the AICC 2012 age scale26 back to 0.8 Ma , where no ice-core dust data exists, to glacials in the LR04 record   Fig.  for that28 that established such correlations for the last glacial cycle between MS and the EDML ice core. However, our records extend the preserved time to the last 25 glacial cycles. We similarly observe strong correlation between MS and Ca counts derived from XRF scanning  suggest that windblown dust may be an important fraction of the total. Thus, it is an open question if MS, Fe and Ca record windblown dust only, or whether they also record terrigenous sediments advected by ocean currents which would have to be controlled ultimately by wind strength, or some combination thereof. Here we make an estimate of windblown dust deposition rates in the Scotia Sea based on published models and focusing factors, and compare this to the terrigenous accumulation rate at Site U1537. Modeled dust flux estimates for the south Scotia Sea31 are ~5 g/m2/year during the LGM. This compares to terrigenous accumulation at U1537 of ~130 g/m2/year during the LGM and 80 g/m2/year in the MIS 6 glacial maximum, where terrigenous accumulation is calculated as total mass accumulation rate (MAR) minus opal MAR  (ref. 23), i.e. 11–29 times more sediment was added to the core site via lateral transport than by vertical settling through the water column. If we assume that the focusing factor at MD77-3134 is 16 and focusing at U1537 is about half that at nearby MD77-3134 , then modeled windblown dust accumulation would be about 8 × 5 g/m2/year = 40 g/m2/year, implying that dust can be a significant fraction of the glacial terrigenous MAR at Site U1537 (30–50% in this example estimate). We emphasize that there is a large degree of uncertainty in modeled dust estimates, for example the only nearby dust observations, at King George Island, are greater than the present day modeled value by an order of magnitude31, and the focusing factor can be extended only very approximately to Site U1537. In contrast to glacial conditions, interglacial dust supply is modeled to be ~0.1 to 1 g/m2/year (refs. 32). The Holocene focusing factor of MD07-3134 is 5–13 (ref. 28), and if, for this calculation, the factor at Site U1537 is half that, 4, then the dust deposition at Site U1537 would be 0.4 to 4 g/m2/year, which is ~1–10% of the Holocene interglacial terrigenous accumulation (~45 g/m2/year), a much lower, and possibly negligible, proportion than for dust deposition in glacials. This back-of-envelope exercise shows that, while there is a large uncertainty, windblown dust can be a significant proportion of the total glacial terrigenous fraction in the southern Scotia Sea.The proportion of terrigenous sediments in Southern Ocean cores that are of windblown origin is difficult to determine because there are few provenance studies and large uncertainties in the dust models, but previous studies  Nd and Pb isotope records from the Atlantic sector of the Southern Ocean suggest absence of Weddell Sea derived Antarctic Bottom Water (AABW) export to the north during the last two glacial maxima10. Also, current speeds were sluggish during the LGM in the northern Weddell Sea, indicative of a slow-down of the Weddell Gyre34. This conclusion is further supported by strong gyre-opposing currents forming contourite drifts in the southeastern Weddell Sea during the LGM35. Since the gyre is primarily wind driven and sea-ice cover was extensive in the Weddell Sea34, circulation was likely much slower or even stalled during glacials, largely prohibiting export of AABW to Site U1537 in the Scotia Sea. During deglaciation, bottom water transport resumed10, current speeds increased by an order of magnitude in the northern Weddell Sea during the Holocene34 and gyre-opposing bottom currents ceased in the southeastern Weddell Sea35, indicating together a declining sea-ice cover and more southerly winds36, and a generally stronger Weddell Gyre, capable of transporting a higher fraction of fine-grained, glaciomarine material to the Scotia Sea during interglacials. We should add that re-deposition of turbidity currents—often a major constituent of sediment drifts—is unlikely to have reached Dove Basin because of the South Orkney Trough between the continent and our sites.Lower glacial and higher interglacial contributions of ocean currents to the terrigenous fraction is also indicated by Weddell Gyre dynamics. Besides the ACC, the Weddell Sea is an important source of Antarctic glaciomarine material that is presently transported to the Scotia Sea through the Orkney Passage37. The contribution of these particles to the MS record depends strongly on their mineralogy. If composed of paramagnetic clay minerals, their contribution to the MS signal would be low. The provenance of terrigenous sediments in the Scotia Sea, using the detrital sedimentary Nd isotopic composition as a tracer, has a more Antarctic (less radiogenic) source during interglacials, and a more Patagonian and Antarctic Peninsula source during glacials39. Dust provenance studies from East Antarctic ice cores indicate Patagonia as the main dust supplier throughout the last 800 ka40. This suggests that windblown dust from Patagonia is a realistic terrigenous source during glacial climates for Dove Basin.Regardless whether terrigenous material is transported by the Weddell Gyre or the ACC, of all grain size fractions present in deep marine sediments, the clay fraction is most efficiently advected within ocean currents41, with sizes extending up to 20 µm, and only minor dust proportions in the <2 µm clay size fraction. Grain sizes >5 µm compose up to 50% of the total dust in the present day at Berkner Island of the Filchner-Rønne Ice Shelf in the southern Weddell Sea42. Trajectory studies identified two Patagonian dust sources with the southernmost area around San Julian’s Great Depression as the dominant dust source43. Dust transport times from Patagonia may be 1 day for our sites31 and up to 7 days for East Antarctic ice cores43. Since the Scotia Sea is located in the trajectory path of atmospheric transport from South America to East Antarctica, there is strong evidence that the eolian record of Site U1537 mostly originated from Patagonia.The median grain size by mass of desert eolian dust today is ~8 µm44, implying that only then were the strongest SHW located over Patagonia. During glacial termination, the wind belt might have shifted south14, leaving Patagonia outside the center of high wind speeds during interglacials. However, the strength and position of the SHW is debated45. In any case, a strong glacial-to-interglacial gradient in dust transport is picked up by all dust proxy records at Site U1537 over each of the last 25 glacial terminations , leaving only 10–20 % of the remaining variability to be driven by independent processes.46, providing more glaciogenic material for both processes during glacials. On millennial time scales, strong atmospheric coupling is mainly observed for glacial maxima and early deglaciations27. During interglacials, this coupling weakens. Sediments from our study site have low dust content because rising sea levels during deglaciations flooded glacial outwash plains, diminished the area of dust availability, and increased the deposition area for terrigenous material on the continental shelves of Patagonia and Antarctica. Also, shifting or weakened SHW might have reduced the dust supply from Patagonia14. We should point out that Fe delivery by icebergs is unlikely a dominant fertilization source in the Scotia Sea because iceberg-rafted debris occurs primarily as pulses during glacial retreat after the LGM and is commonly lower during both glacials and interglacials in Iceberg Alley20.The close correlation of dust proxies MS, Fe and Ca with Antarctic glacial dust indicates that oceanic processes (Weddell Gyre and ACC dynamics) and atmospheric processes (SHW dynamics and the hydrologic cycle) are coupled on orbital and millennial scales. Interhemispheric ice-sheet ocean connections were coupled on orbital time scales mainly through sea-level forcing47. Patagonia is the major dust source for the sites discussed here in the Atlantic sector of the Southern Ocean and Antarctic continent, i.e. IODP Site U1537, ODP Site 1090 and ice core EDC . The remaining dust sources in Southern Africa and Australia (data from18) are mainly relevant for the Indian and Pacific Ocean, respectively  change obtained in the Mediterranean50. The MPT has hitherto not been recovered continuously in Antarctic ice cores; however, suitable locations have been recently found in the interior of East Antarctica to drill ice as old as 1.5 Ma. Across the MPT, global climate cooled  ago) at high resolution in the AZ. This is a critical period when the dominant glacial-interglacial cyclicity shifted from a 41-kyr to a 100-kyr periodicity Fig. , despiteed Figs.  and 5 and51 Fig. . Togethe6. Our dust proxy records for Site U1537 exhibit almost exclusively 41-kyr power before ~1.25 Ma  providing a larger and more glaciated Patagonian dust source. It could also imply that glacial-to-interglacial latitudinal shifts or intensity variations in SHW or the hydrological cycle were more important in the western South Atlantic close to the dust source than amplitude changes in sea level and ice volume before and throughout the MPT, whereas in the SAZ dust deposition increased along with global ice volume during the MPT , at Site U1537  and that 230Th-normalization method works reliably only over ~the last two glacial cycles because of the 230Th half-life, conversion of the long U1537 dust and ocean productivity time series to flux data  might shed light on sediment focusing in future studies. However, even if converted to fluxes, the dust proxies maintain their high-amplitude, glacial-to-interglacial variability and match the amplitude changes in ice cores, regardless . All in all, our data clearly indicate enhanced surface ocean productivity during interglacials  almost through the entire core section studied  shows a clear out-of-phase relationship with higher biogenic opal productivity, demonstrating that sea-ice reduces productivity in this setting. In contrast, greater primary productivity is promoted by lower sea-ice coverage, as demonstrated by elevated opal contents and low IPSO25. The latter is further supported by analysis of a suite of phytosterols . However, this site is located 2900 km farther east, in the open AZ of the Southern Ocean and north of the maximum winter sea-ice extent   Fig. . Under g61) Fig. , atmosph63 with only limited injection of nutrients from deep to surface waters, thereby reducing glacial productivity4. Further, the glacial-interglacial amplitude in atmospheric CO2 varies widely (up to 100 ppm) throughout the last 1.5 Ma  in the AZ. Instead, the net CO2 drawdown during glacials could have resulted from more efficient trapping of CO2 in the deep ocean by a combination of spatially expanded sea ice, longer mean deep water residence times in the deep sea, and enhanced surface-ocean stratification in the AZ, and active CO2 drawdown by increased productivity in the SAZ7. Our data support such a view.As such, biosiliceous productivity in the Scotia Sea appears insensitive to the increase in glacial Fe supply from dust, or may lack other micro-nutrients and was thus unlikely responsible for significant glacial drawdown in atmospheric COThe glacial-interglacial pattern of inferred productivity changes generally follows global ice volume with an asymmetrical pacing of relatively fast changes from low to high ocean productivity at the begin of an interglacial, and a slow decline during warm periods when compared to the LR04 stack  Figs.  and 7. H23  is considered one of the so-called “superinterglacials” in the Northern Hemisphere and around Antarctica Ma is co66) became progressively colder between MIS 19 and 11 (~0.43 Ma)  all show increased opal amplitudes with plateau-like peaks  until ~0.47 Ma. Sea-surface temperatures at Site ODP 1094  all reach plateau-like highs and exhibit the largest glacial-to-interglacial amplitude in productivity changes. This is likely related to changes associated with the MBE, a marked global increase in climate variance during MIS 12 at ~0.43 Ma. Since MIS 12, ice-cores document interglacials with higher atmospheric COels Fig. , while δ2 drawdown by siliceous primary producers from the atmosphere through Fe fertilization in the high latitude AZ. Instead, we see clear evidence for spatially and temporally variable controls of ocean productivity and dust deposition across the Southern Ocean. Further, marine terrigenous deposition depended on the extent of bottom water export and Weddell Gyre dynamics.Overall, we see a distinct increase in dust deposition during glacials but little indication for enhanced glacial COIn Iceberg Alley, ocean productivity increased during interglacials, especially during superproductivity periods before the MPT and after the MBE. We also identify a close relationship between dust proxies in Iceberg Alley and variations in global ice volume and sea level, both of which are driven mainly by ice-sheet size variations in the Northern Hemisphere. However, glacial dust deposition was high in the AZ throughout the last 1.5 Ma, while it increased in the SAZ during the MPT, indicative of increasingly stronger and farther reaching SHW or less regional differences in the hydrological cycle. Half way through the MPT, around 0.9 Ma, the coupling between global ice volume and dust became very strong and the glacial-to-interglacial sea-level amplitudes increased. Accordingly, we see a close interhemispheric coupling of cryospheric, oceanic and atmospheric processes in the AZ throughout the last 1.5 Ma.2, with northward movement of the sea-ice front and SHW during glacials reducing gas release from the ocean to the atmosphere across the AZ, and southward movement of the sea-ice front and SHW associated with higher upwelling and paleoproductivity as well as enhanced CO2 release to the atmosphere during interglacials, especially during superinterglacials. Also, reduction in glacial atmospheric CO2 but no additional CO2 drawdown through the MPT is consistent with our records.The Scotia Sea records also highlight that paleoproductivity, SHW and sea-ice feedbacks play a crucial role in regulating atmospheric CO70. Site MD07-3134 lies ~40 km southwest on the same contourite in 3663 m water depth and is located at 59° 25’ S, 41° 28’ W with a core length of 58.2 m (ref. 27). For this study we use the two longer holes that were drilled at Site U1537 with the Advanced Piston Corer. At Hole U1537A the upper 264.0 meters below seafloor (CSF-A) were drilled with a recovery of 268.9 m (102%). At Hole U1537D the upper 354.3 m CSF-A were drilled and recovered 349.0 m (99%). Both holes were retrieved during calm weather conditions and obtained virtually the same sedimentary record with major lithologic features occurring at similar depths in each hole  Site U1537 is located in the center of Iceberg Alley in the southern Scotia Sea . Hence the start of our reconstruction at 1.5 Ma translates into ~183 m (CSF-A) in Hole U1537A, ~182 m (CSF-A) in Hole U1537D, and ~195 m (CCSF) for the splice of Site U1537 (ref. 16). All depths reported in this paper are in m CCSF.Our scientific objectives require recovery of complete stratigraphic sections to the best extent possible to establish robust age controls Fig. . Such a The core sections were first analyzed onboard JOIDES Resolution with the Whole-Round Multisensor Logger at 2-cm intervals to determine MS Figs.  and 4 wi72.After core splitting, the Section Half Multisensor Logger (SHMSL) was used to measure MS again . Results for each element are given as peak area intensities determined in total counts per second (cps). The measurement increment varied between 2 cm and 1 mm.Postcruise, we also measured the distribution of chemical elements using an AVAATECH X-ray Fluorescence (XRF) Core Scanner along the splice of Site U1537  detector, a KBr beam splitter, and a HTS-XT accessory unit. Each sample was scanned 64 times at a wavenumber resolution of 4 cm−1  for the wavenumber range from 520 cm−1 to 3750 cm−1 with the aperture set to 8 mm in diffuse reflectance mode.Fourier Transform Infrared Spectroscopy (FTIRS) was used to quantify biogenic opal to obtain information on past changes in ocean productivity. We measured 616 samples covering the last 1.4 Ma Fig. , of whic28, which has been shown to produce reliable results with an accuracy as determined by means of the root mean square error of cross-validation of 4.7 % (in % bSi). The absolute percentage error (MAPE) would be ≤11% over the entire 0–100% opal range and ≤5.7% above 10% opal74, which is primarily an effect of scaling of the relative error of the calibration. Comparison of laboratory standards measured by means of the different wet chemical and the FTIRS method yielded very similar results74. Precision and reproducibility of the FTIRS method is high for the U1537A samples batch with a mean deviation of 3.4% and a range of 0–8.4% of the measured value.A partial least squares regression model was used based on synthetic sediment samples with known biogenic opal content28.FTIRS has been implemented successfully to reconstruct paleoproductivity on neighboring cores MD07-3133 and MD07-3134 for which FTIRS derived opal has been compared against opal measured by means of “conventional” leaching techniques75 was carried with a slight modification to the extraction method. Briefly, freeze-dried samples (ca. 3–4 g) were saponified in a methanolic Potassium Hydroxide (KOH) solution ; 5 % KOH) for 60 min (70 °C). Hexane (3 × 2 ml) was added to the cooled saponified content, with supernatant containing non-saponifiable lipids (NSLs) transferred to clean vials and dried over Nitrogen . NSLs were then further fractionated using silica  column chromatography. HBIs were eluted with hexane (6 ml) and purified further using silver-ion chromatography , with saturated compounds eluted with hexane (2 ml) and unsaturated compounds containing HBIs collected in a subsequent acetone fraction (3 ml). Sterols were collected of silica columns with Hexane:Methyl Acetate, (4:1 (v/v), 6 ml). Prior to extraction, samples were spiked with an internal standards 9-octylheptadec-8-ene (9–OHD) and 5α-androstan-3β-ol (100 ng each) to permit quantification of HBIs and sterols, respectively.Lipid analysis75. Identification of individual biomarkers was based on their characteristic retention indices and mass spectra, while quantification was achieved by comparison of mass spectral responses of selected ions with those of internal standards, and normalized according to their respected instrumental response factors and the mass of sediment extracted75. Prior to analysis, sterol-containing fractions were derivatised with N,O–bis(trimethylsilyl)trifluoroacetamide .Analysis of HBIs and sterols was carried out using an Agilent 7890 A GC coupled to a 5975 series mass selective detector and operating conditions76). We used the five magnetic reversals  as first order age control points. In our age modeling, we used the depth range, defined by the last and first occurrence of stable polarity below and above the transition interval. The reversals were identified in both Holes U1537A and U1537D at almost identical depth . The final, resulting age model for Site U1537 consists of 151 tie points  shows only one major change around the MBE from ~10 cm/kyr before to ~20 cm/kyr after that time.The high-resolution age models rely on tuning MS maxima of Site U1537 to dust maxima of the EDC ice core20 show comparable temporal variability and would produce very similar high-resolution age models . Three types of age-control points were used 79, particularly for the age of the upper boundary of the Cobb Mountain Subchron, which is 15 kyr younger on the 2012 GPTS than the LR04 timescale78.230Th-normalization data for neighboring Site MD07-3134 indicates interglacial focusing factors of 5–13 and glacial focusing factors of 11–29 (ref. 28). Due to the radioactive decay, this method is only applicable over ~the last two glacial cycles. Hence, calculation of MAR on our long time series is problematic and will likely lead to erroneous results and misinterpretations.Concentration profiles for proxies used in this study  demonstrate clear glacial to interglacial trends. Do these trends hold if converted, as far as possible, into flux rates? To answer this question one needs to consider that the substantially elevated accumulation rates in the Scotia Sea relative to surrounding areas are achieved through syn-sedimentary focusing of material within Weddell Sea sourced AABW and the overlying ACC. 72 to determine DBD for each non-destructive GRA measurement  are described as silty-clay with >25% quartz and <10% opaque minerals.Accordingly, interglacial periods show high opal contents, up to 55–60 %, especially in the upper part of the U1537 record, whereas glacial periods generally range 0–10 %. The relationship is striking for Site U1537 and holds for the entire record. We emphasize that intervals of elevated estimated opal content are found in all lithostratigraphic descriptions as thick sedimentary packages of diatom oozes with >50% diatoms and <10% spicules as identified in smear slides28). GRA values below ~1.3 g/cm3 can only be found in unconsolidated sediments of high porosity and low grain density80—conditions usually only met if marine deposits are rich in biogenic opal , those sections are exclusively diatom oozes. The temporal variability of GRA at Site U1537 shows such systemically low values only during interglacials, whereas glacial deposits are ≥1.6 g/cm3; providing further indication for substantially higher opal contents during interglacials. We should note, however, that we did not attempt to estimate biogenic opal quantitatively from GRA since there is a well-known general down-core trend toward higher values due to increasing compaction and de-watering of the sediment, especially within the upper 120 m  has also been used to estimate opal content in neighboring Site MD07-3134 ref. 8. GRA va16 did not show significant preservation changes in the section presented here. More severely, diagenetic changes could occur deeper in the sediment column, near the opal C/T boundary, which is often in 400–600 m water depth in the circum-Antarctic realm. However, we only report data from the top 200 m . Also, the fact that productivity proxies Si/Al and Si/Ti show the same long-term step changes argues against increasingly poor opal preservation downcore. In addition, the smear slides investigated onboard JRThe fact that the glacial-to-interglacial pattern is mainly driven by climate-related changes producing opal-rich and terrigenous-rich deposits, is also shown by the variability of NGR. Higher values generally indicate elevated content of radioactive isotopes of potassium (K) thorium (Th) and uranium (U), which are commonly enriched in clay minerals. XRF scanning data also shows that specifically K, Fe, and Ti are elevated for clay-rich sediment and restricted to glacial periods. Hence, clays dominate glacial times at Site U1537 and their high NGR is inversely correlated to GRA. The systematic, high-amplitude glacial-to-interglacial variability is again striking and holds through the entire record Fig. . In summSupplementary Information