Oral case presentations – structured verbal reports of clinical cases – are fundamental to patient care and learner education. Despite their continued importance in a modernized medical landscape, their structure has remained largely unchanged since the 1960s, based on the traditional Subjective, Objective, Assessment, Plan (SOAP) format developed for medical records. We developed a problem-based alternative known as Events, Assessment, Plan (EAP) to understand the perceived efficacy of EAP compared to SOAP among learners.We surveyed  all third- and fourth-year medical students and internal medicine residents at a large, academic, tertiary care hospital and associated Veterans Affairs medical center. The primary outcome was trainee preference in oral case presentation format. The secondary outcome was comparing EAP and SOAP on 10 functionality domains assessed via a 5-point Likert scale. We used descriptive statistics (proportion and mean) to describe the results.n = 41) preferred the EAP format as compared to 19% (n = 11) who preferred SOAP (p < 0.001). EAP outperformed SOAP in 8 out of 10 of the domains assessed, including advancing patient care, learning from patients, and time efficiency.The response rate was 21% (118/563). Of the 59 respondents with exposure to both the EAP and SOAP formats, 69% (Our findings suggest that trainees prefer the EAP format over SOAP and that EAP may facilitate clearer and more efficient communication on rounds, which in turn may enhance patient care and learner education. A broader, multi-center study of the EAP oral case presentation will help to better understand preferences, outcomes, and barriers to implementation.The online version contains supplementary material available at 10.1186/s12909-023-04292-3. Excellent inter-physician communication is fundamental to both providing high-quality patient care and promoting learner education , and hasGiven that the goals of a medical record are different than those of oral case presentations, it should not be assumed that they should share the same format. While Dr. Weed sought to make the medical record as “complete as possible,”  internalIn accordance with Dr. Weed’s “problem-oriented approach to data organization,”  but withEAP is a problem-based format used at the discretion of the attending physician. In line with suggested best practices , the EAPWe surveyed third- and fourth-year medical students, and first- through fourth-year internal medicine and internal medicine-pediatrics residents, caring for patients at a large, academic, tertiary care hospital and an affiliated Veterans Affairs medical center. Internal medicine is a 12-week core clerkship for all medical students in their second year, with 8 weeks spent on the inpatient wards. All student participants had completed their internal medicine clerkship rotation at the time of the survey. We did not conduct a sample size calculation at the outset of this study.An anonymous, electronic survey  was created to assess student and resident experience with and preference between EAP and SOAP oral case presentation formats during inpatient internal medicine rounds . For comparative analysis between EAP and SOAP, responses from respondents who had experience with both formats were compared using the Wilcoxon Signed Rank Test to evaluate differences. All statistical analyses were done using SAS V9.4 . We considered n = 62/441) among medical students and 46% (n = 56/122) among residents. Respondents were 61% (n = 72) female. A total of 98% (n = 116) and 52% (n = 61) of respondents reported experience with SOAP and EAP formats, respectively. Among medical students, 60% (n = 37) reported experience with SOAP only while 39% (n = 24) had experience with both formats. Among residents, 36% (n = 20) and 63% (n = 35) had experience with SOAP only and both formats, respectively . The response rate was 14% (n = 41) preferred the EAP format as compared to 19% (n = 11) preferring SOAP (p < 0.001). The remainder  indicated either no preference between formats or indicated another preference. Among residents, 66% (n = 23) favored EAP, whereas 20% (n = 7) and 14% (n = 5) preferred SOAP or had no preference, respectively (p < 0.001). Among students, 75% (n = 18) favored EAP, whereas 17% (n = 4) and 8% (n = 2) favored SOAP or had no preference, respectively (p < 0.001).Of the 59 respondents with exposure to both the SOAP and EAP formats, 69% (p < 0.001) and encouragement to: focus on assessment and plan , distill pertinent information , and integrate data  (Table p < 0.001), its capacity to convey one’s thinking , and its ability to facilitate learning from peers  and one’s own patients . There were no significant differences in the amount of time allotted for discussing the patient’s subjective experience or in ease of use.Likert scale ratings for domains assessed by trainees who had experience in either format are shown in Table Evaluation of trainee free text responses regarding oral case presentation preference revealed several general themes Table . First, Our single site survey comparing 2 oral case presentation formats revealed a preference among respondents for EAP over SOAP for those medical students and internal medicine residents who had experience with both formats. Furthermore, EAP outperformed SOAP in 8 out of 10 of the functionality domains assessed, including areas such as advancing patient care, learning from patients, and, particularly, time efficiency. Such a constellation of findings implies that EAP may not only be a more effective means to accomplish the key goals of oral case presentations, but it may also provide an opportunity to save time in the process. In line with SOAP’s current de facto status as an oral case presentation format, almost all respondents reported exposure to the SOAP format. Still, indicative of EAP’s growing presence at our academic system, more than one third of medical students and more than one half of residents also reported having experience with the EAP format.While limited data exist that compare alternative oral case presentations to SOAP on inpatient medicine rounds, such alternatives have been previously trialed in other clinical venues. One such format, the multiple mini-SOAP, developed for complex outpatient visits, encourages each problem to be addressed “in its entirety” before presenting subsequent problems, and emphasizes prioritization by problem pertinency . The creAnother approach has been assessed in the emergency department. Specifically, Maddow and colleagues explored assessment-oriented oral case presentations to increase efficiency in communication between residents and faculty at the University of Chicago . In the Prior to our study, the nature of the format for inpatient medicine oral case presentations had thus far escaped scrutiny. This is despite the fact that oral case presentations are time (and therefore resource) intensive, and that they play an integral role in patient care and learner education. Our study demonstrates that learners favor the EAP format, which has the potential to increase both the effectiveness and efficiency of rounding.Still, it should be noted that a transition to EAP does present challenges. Implementing this problem-based presentation format requires a conscious effort to ensure a continued holistic approach to patient care: active problems should be defined and addressed in accordance with patient preferences, and the patient’s subjective experience should be meaningfully incorporated into the assessment and plan for each problem. During initial implementation, attending physicians and learners must internalize this new format, often through trial and error.From there, on an ongoing basis, EAP may require more upfront preparation by attending physicians as compared to SOAP. While chart review by attendings in advance of rounding is useful regardless of the format utilized, this practice is especially important for the EAP format, where trainees are empowered to interpret and distill – rather than simply report a complete set of – information. Therefore, the attending physician must be aware of pertinent data prior to rounds to ensure that key information is not neglected. Specifically, attendings should pre-orient themselves with laboratory values, imaging, and other studies completed, and new suggestions from consultants. More extensive pre-work may be required if teams wish to employ the EAP format for newly admitted patients, as attending physicians must also familiarize themselves with a patient’s medical history and their current presentation prior to initial team rounds.Our findings should be interpreted within the context of specific limitations. First, low response rates may have led to selection bias within our surveyed population. For instance, learners who desired change in the oral case presentation format may have been more motivated to engage with our survey. Second, there could be unmeasured confounding variables that could have skewed our results in favor of the EAP format. For example, attendings who utilized the EAP format may have been more likely to innovate in other ways to create a more positive experience for learners, which may have influenced the scoring of the oral case presentation format. Third, our findings were largely based on subjective experience. Objective measurement  may lend additional credibility to our findings. Lastly, our study included only a single site, limiting our ability to generalize our findings.Our study also had several strengths. Our learner participant pool was broad and included all third- and fourth-year medical students and all internal medicine residents at a major academic hospital. Participation was encouraged regardless of the nature of a participant’s prior exposure to different oral case presentation formats. Our survey was anonymous with randomization to mitigate order bias, and we focused our comparison analysis on those who had exposure to both the EAP and SOAP formats. We collected data to compare EAP with SOAP in 2 distinct ways: head-to-head preference and numeric ratings amongst key domains. Both of these methods demonstrated a significant preference for EAP among learners in aggregate, as well as for students and residents analyzed independently.Our findings suggest a preference for the EAP format over SOAP, and that EAP may facilitate clearer and more efficient communication on rounds. These improvements may in turn enhance patient care and learner education. While our preliminary data are compelling, a broader, multi-center study of the EAP oral case presentation is necessary to better understand preferences, outcomes, and barriers to implementation. Further studies should seek to improve response rates, for the data to represent a larger proportion of trainees. One potential strategy to improve response rates among medical students and residents is to survey them directly at the end of each internal medicine clerkship period or rotation, respectively. Ultimately, EAP may prove to be a much-needed update to the “currency with which clinicians communicate.”Additional file 1: Appendix A. Exemplar Transcripts .Additional file 2: Appendix B. Survey Instrument.


Mus musculus (MmGSTP1-1). The lead compound was selected following the screening of a library of currently approved and registered pesticides that belong to different chemical classes. The results showed that the fungicide iprodione  exhibited the highest inhibition potency (ΙC50 = 11.3 ± 0.5 μΜ) towards MmGSTP1-1. Kinetics analysis revealed that iprodione functions as a mixed-type inhibitor towards glutathione (GSH) and non-competitive inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). X-ray crystallography was used to determine the crystal structure of MmGSTP1-1 at 1.28 Å resolution as a complex with S-(p-nitrobenzyl)glutathione (Nb-GSH). The crystal structure was used to map the ligand-binding site of MmGSTP1-1 and to provide structural data of the interaction of the enzyme with iprodione using molecular docking. The results of this study shed light on the inhibition mechanism of MmGSTP1-1 and provide a new compound as a potential lead structure for future drug/inhibitor development.Multidrug resistance is a significant barrier that makes anticancer therapies less effective. Glutathione transferases (GSTs) are involved in multidrug resistance mechanisms and play a significant part in the metabolism of alkylating anticancer drugs. The purpose of this study was to screen and select a lead compound with high inhibitory potency against the isoenzyme GSTP1-1 from  A large superfamily of enzymes known as glutathione transferases GSTs; EC 2.5.1.18) catalyze the nucleophilic attack of the reduced tripeptide glutathione  on nonpolar molecules that contain an electrophilic carbon, oxygen, or sulfur atom .5.1.18 c. GSTs plGSTs are primarily classified based on their structure and sequence . They arGSTs function as dimers consisting of two similar or different subunits, each with an average sequence length of 200–250 amino acids. Each subunit of GSTs has at least two binding sites, the G-site and the H-site ,5,7, whiThe catalytic role of GSTs in multidrug resistance (MDR) of cancer cells is mainly due to their overexpression, leading to rapid detoxification of anticancer drugs ,11,12. TA range of synthetic and natural substances has been tested for their ability to inhibit GSTs in an attempt to lessen or even abolish multidrug resistance in cancer cells. 2,2′-dihydroxybenzophenones , benzoxa50 values in the low micromolar range. Recently, Bodourian et al. , iprodione was selected for further studies. The results showed that the insecticide deltamethrin and the fungicide iprodione display the highest inhibition potencies, 82% and 78%, respectively. Considering that among deltamethrin and iprodione, the latter displays much less toxicity [e.g., acute oral toxicity LDOther pesticides with significant but much lower inhibition potency compared to iprodione, include spiromesifen (70% inhibition), α-endosulfan (58% inhibition), and fenamidone (53% inhibition). 50) which was 11.3 ± 0.5 μM (R2 = 0.989)  . This vaA kinetic inhibition study was performed with the aim of determining the inhibition pattern of iprodione with the enzyme, and the results are illustrated in iE ≠ KiES). The analysis reveals that the inhibition constants KiE and KiEGSH for iprodione, using GSH as a variable substrate, are 5.2 ± 0.1 μM (R2 = 0.99) and 14.8 ± 4 μM (R2 = 0.99), respectively. The mixed-type inhibitor indicates that iprodione can bind to both the free enzyme and the enzyme–substrate complex (E-GSH) with different affinities for each enzyme  suggest that iprodione shows preferential binding and greater affinity for the free form of MmGSTP1-1. This suggests that the recognition elements of binding for the inhibitor are not completely overlapping with those of the substrate, and therefore, the substrate and inhibitor binding sites may be thought to be distinct. Thus, the binding of iprodione to MmGSTP1-1 may not preclude GSH binding. The non-competitive inhibition constant using CDNB as a variable substrate is Ki = 6.6 ± 2.6 μM (R2 = 0.98). The measured KIt is well established that the inhibition pattern that is obeyed by different GST isoenzymes when studied with different inhibitory compounds is inhibitor- and substrate-dependent and cannot be predicted based on structural information of the particular inhibitor or the enzyme. However, the vast majority of kinetic inhibition studies have revealed that the most potent inhibitors function as non-competitive/mixed-type inhibitors ,27,32,33MmGSTP1-1 was refined to 1.28 Å resolution with final Rwork and Rfree values of 0.175 and 0.199, respectively  [The crystal structure of ectively . MmGSTP1olution)  which haMmGSTP1-1 structure at a lower resolution (1.8 Å) [A molecule of Nb-GSH was found bound in the active site of each molecule of the dimer . A struc (1.8 Å)  showed aMmGSTP1-1  and using CDNB as a variable substrate (Ki = 6.6 ± 2.6 μM), supporting the accuracy of the in silico docking studies.Based on the estimation made by AutoDock 4.2, the free energy of binding is equal to −7.17 kcal/mol and the inhibition constant KMmGSTP1-1.The discovery of GST inhibitors remains a challenging task . This isMmGSTP1-1 in complex with Nb-GSH and molecular docking was used to investigate the interaction between MmGSTP1-1 and iprodione.Iprodione acts as a mixed-type inhibitor towards GSH and as a non-competitive inhibitor towards CDNB. X-ray crystallography was used to determine the crystal structure of Iprodione exhibits minimal toxicity to mammals and, therefore, can be considered as a new lead compound that can be further investigated. Repurposing already approved drugs seems to be a very attractive approach given the investment  required for implementing a new drug discovery project. In this way, the drug development timeline can be shortened since several existing compounds already have known technology, safety, and toxicity profiles.


Here, we explore Miocene ice-ocean-climate interactions by presenting a multi-proxy reconstruction of subtropical front migration, bottom water temperature and global ice volume change, using dinoflagellate cyst biogeography, benthic foraminiferal clumped isotopes from offshore Tasmania. We report an equatorward frontal migration and strengthening, concurrent with surface and deep ocean cooling but absence of ice volume change in the mid–late-Miocene. To reconcile these counterintuitive findings, we argue based on new ice sheet modelling that the Antarctic ice sheet progressively lowered in height while expanding seawards, to maintain a stable volume. This can be achieved with rigorous intervention in model precipitation regimes on Antarctica and ice-induced ocean cooling and requires rethinking the interactions between ice, ocean and climate.Gradual climate cooling and CO Hou et al. propose, based on dinocysts, clumped isotopes and ice sheet modelling, that during Miocene cooling, the Antarctic ice sheet progressively lowered in height while expanding seawards, to maintain a relatively stable volume. Variability in the latitudinal sea surface temperature (SST) gradient is mostly a function of polar temperatures, which are much more variable than those at low latitudes because of polar amplification3. In turn, polar SSTs, especially offshore Antarctica, vary with prevailing cryosphere conditions, including sea ice extent5. The steepest part of the latitudinal SST gradient is at mid-latitudes, at the boundary between subtropical gyres and subpolar waters. In the Southern Hemisphere, this is the subtropical front (STF): the northern limit of the Southern Ocean and the Antarctic Circumpolar Current (ACC), and the centre of ocean carbon uptake6  was as high as projected for the future.Temperature contrasts between the equator and high latitudes are mitigated through poleward atmospheric and ocean heat transportke6 Fig. . The ACCpCO2 declined from 800 to 300 parts per million (ppm)17, global temperatures dropped19, latitudinal SST gradients increased20 and global ice volume23 and sea ice expanded24. The current paradigm assigns pCO2 decline as the primary driver, which, through polar amplification of cooling, stimulates ice growth and cooling in the regions of deep-water formation26. Yet, recent data have challenged this view. A recent study found that Neogene SST gradients increased in the subtropical gyre but decreased from the subtropical front to polar waters27. With relatively stable equatorial29 and polar SSTs30, this indicates that the mid-latitudes, rather than the high-latitudes27, cooled most profoundly in the Neogene. Antarctic-proximal records suggest a retreated Antarctic ice sheet and warm Antarctic-proximal conditions31 during the mid-Miocene Climatic Optimum (MCO) and profound seaward ice sheet advance during subsequent cooling termed the Middle Miocene Climatic Transition (MMCT)34, in line with pCO2 estimates35. Along with a rise in deep ocean benthic foraminifera oxygen isotope ratios (δ18Obf), this suggests a strong increase in global ice volume. Yet, the existing series of clumped isotope measurements  on Miocene benthic foraminifera38 suggest higher-than-previously-estimated Bottom Water Temperatures (BWTs) during the MCO, and as a result, large global ice volume. These records also indicate strong BWT cooling during the MMCT, explaining most if not all of the δ18Obf rise, and therefore little to no ice volume buildup. How this generally connects to far-field sea level changes is still poorly reconciled. However, the uncertainties in clumped isotope data and the limited resolution and temporal range of the records leave ambiguity on the true amount of BWT drop and ice volume buildup during the mid-Miocene.Throughout the Neogene (23–2.58 Ma), 39, intensified in the late Oligocene ~26 Ma40 but modern-like strengths only developed in the late Neogene41. The development and latitudinal position of the fronts associated to the ACC are, however, still poorly constrained. Meanwhile, a long-term trend of BWT change and how the oceanic processes are coupled to Antarctic ice dynamic is still unclear. To shed light on the links between (Antarctic) ice volume and dynamics, Southern Ocean oceanography and latitudinal SST gradients, we present a detailed reconstruction of Neogene STF migration history and surface and bottom water temperature offshore Tasmania, and pair these with estimates of Antarctic ice volume change from the MCO across the MMCT. We use dinoflagellate cyst (dinocyst) biogeography42 to reconstruct the position of Southern Ocean currents and fronts and combine these with published SST reconstructions27. Finally, from benthic foraminiferal Δ47, we assess deep-water temperature changes at Ocean Drilling Program (ODP) Site 1168, as well as sea water δ18O (δ18Osw) as a proxy for Antarctic ice change.Like the modern, changes in the Southern Ocean, notably regarding fronts and currents, were likely vital for heat transport towards the ice sheet in the Neogene. A relatively weak ACC, initiated during the Eocene18O evolution, implying stable global ice volume. After 7 Ma, the northward shift of the STF is limited by the Australian continent, even though the SSTs continue to decrease. To reconcile expansion of subpolar ocean conditions and progressive Neogene Southern Ocean cooling with stable ice volume and compelling evidence of ice advance, we argue that the Miocene Antarctic ice sheet progressively lowered in height while expanding seawards during the mid-Miocene. We present idealised ice sheet model simulations that physically constrain this hypothesis. This changed geometry induced strong regional oceanographic responses with expansion of sea ice, cooling of the region of bottom-water formation and northwards migration of ocean fronts.In this study, we demonstrate that there is a strengthening and equatorward migration of the STF from ~53° to ~42° between ~14 Ma and 7 Ma, concurrent with progressive sea surface and bottom water cooling. The deep ocean cooling can completely explain benthic foraminifer δ42 (see “Methods” section) hence allows reliable reconstructions of paleoceanographic conditions.The vast majority of the dinocysts encountered in the Neogene sediments from ODP Site 1168 are extant species of the modern Southern Ocean. The use of inferences from modern biogeographic distributions and affinities of dinocyst assemblage clustersSpiniferites spp.  Leeuwin Current, delivering heat and sustaining low-latitude dinoflagellate assemblages from western Australia towards the site. It implies that the STF was located to the south of the site. Gradual increases of Operculodinium spp. in this interval suggests gradually cooler-water influence, with an approaching STF from the south. We find occasional northward migrations of the STF  in sporadic abundance of N. labyrinthus, concomitant to SST cooling  is found most abundant in sediments south of the STF, in the modern subantarctic zone. We interpret the proliferation of Nlab and a progressive cooling towards subantarctic zone-like conditions  marks the first interval of prevailing hus Fig. . This sp 48 Fig. . Subsequ 48 Fig. , indicat 48 Fig.  variabilN. labyrinthus started to decrease in abundance. We interpret a southward migration of the frontal systems relative to Australia from the decline in Nlab and the return of north-of-STF clusters. Apparently, this continued cooling is not directly related to continued shifts of ocean frontal systems, but a cooling of the STF itself and tectonic drift. The Nlab-Cluster and the slightly warmer high-Ocen-Cluster alternate in the Pliocene (4.5–2.5 Ma) on orbital timescales  Leeuwin Current at the Site  to 2.5‰, followed by a further rise to ~2.8‰ at the end of the Miocene (~5.3 Ma). Remarkably, we do not record pronounced steps across the MMCT as seen in other records49. The pronounced δ13Cbf maxima (from 17 Ma) likely reflects the Monterey carbon isotope excursion52 and values are in line with those in other records.The δs37 Fig. , includi38 and thus Antarctic ice dynamics  in the MCO (17–14.5 Ma) to 5.0 ± 2.5 °C around 10–9 Ma  and late Miocene .Previous studies have pointed out the unexpected warmth of mid-Miocene BWTs in their reconstructions and discussed potential but undiscernible biases on Δd Figs. S and S3, 18Osw values from Site 1168 BWTs are 0.3 ± 0.5‰ throughout the MCO and MMCT until 9 Ma  from 16 to 9 Ma could in principle be all reconciled with the ~5 °C BWT drop we infer from the clumped isotope data  may allow for some ice volume change (0.3 ± 0.5‰). Given the very similar MCO BWTs derived from multiple sites globally37 we deem the average MCO BWT of 9.9 °C reliable. By binning our MCO data with that of other sites, the uncertainty in that interval can be further reduced to ±1 °C. The 5 ± 2.5 °C BWT in the 10–9 Ma interval is based on most replicates, and thus has the smallest uncertainty. Only when the 10–9 Ma BWT is at the high end of its 95% confidence interval, can the global 25 m RSL (Relative Sea Level) ice volume build-up of Rohling et al.19 be replicated with our Δ47 data. Apart from δ18O-based reconstructions, local eustatic reconstructions of the mid and late Miocene are relatively crude and scarce59, and inevitably hindered by local tectonic activities61. Nevertheless, the inconsistence between Δ47-based absolute δ18Osw (therefore ice volume) and far-field sea level reconstructions is a general arising question dependent on calibrations62 and requires further exploration64. Given the low probability of that scenario, we conclude that the clumped isotope data imply a stronger cooling and thus less ice volume build-up during MMCT than in the model of Rohling et al.19.The calculated δ Ma Fig. . The incata Fig. . Thus, o47-based BWT reconstructions, the higher-than-modern δ18Osw for the mid-Miocene  seems difficult to reconcile with evidence for Antarctic-proximal sea surface warmth30, Mg/Ca-based deep-sea warmth65 and high pCO267 during the MCO. The relatively stable long-term δ18Osw trend  in southwest Australia81, both interpreted as a southerly shift in the frontal systems and westerlies relative to the Australian continent. At the same time, at the Agulhas Plateau82 and in the South Atlantic83, oceanographic reconstructions suggest an equatorward migration of oceanic fronts, rather than a southward migration as in Australia. This suggests an asymmetric behaviour of oceanic fronts around Antarctica.The dinocyst assemblages, combined with previously published SST reconstructions18Obf of 0.2‰ between ~9 and ~6 Ma to about 20 m RSL-equivalent global ice volume build-up  seem to lack global ice volume increase, while time intervals with relatively stable pCO2 (late Miocene) seem to have profound ice volume growth, suggesting a large role for non-linear feedbacks. These fundamental observations put a perspective on the way radiative forcing and complex feedbacks in ocean-ice-atmosphere interactions shaped Neogene ice volume and global climate trends.Taking the above together, the available data show little evidence for Miocene ice volume increase forced by CO92. The site sits on the northern edge of the Subtropical Convergence zone, which separates warm, saline subtropical waters from comparably cold and fresh subantarctic water masses93. During the Neogene, the location of Site 1168 tectonically drifted along with Tasmania and Australia from 52°S at 23 Ma to its modern position at 42 °S94. The Neogene bathymetry was lower bathyal/upper abyssal (1000–2500 m), midway on the continental slope92. During this northward tectonic drift, the Southern margin of Australia was continuously bathed by the eastward flowing (proto-) Leeuwin Current95. Hence, Site 1168 is well-suited to study the Neogene evolution of the STF. We applied the same age model for the sediments as in Hou et al.27.   before they were dissolved with 30% hydrochloric acid (HCl) and 38% hydrofluoric acid (HF) for carbonate and silicate removal, respectively. The remaining palynological residues were sieved on a 10 μm nylon mesh, using an ultrasonic bath to disintegrate agglutinated organic particles. The palynological residues were mounted on glass slides using glycerine, sealed, and counted (under 200 and 400 magnification) using an Olympus CX41 optical microscope. When possible, at least 200 dinocyst specimens were counted98. Samples containing less than (including) 50 dinocyst specimens were excluded for further analysis and interpretation.We studied 131 samples for palynological content42.  to remove adhering sediment, organic lining and nannofossils. The test fragments were dried at room temperature overnight. In order to obtain enough material, other benthic species are also processed. We use Cibicidoides mundulus and Cibicidoides (Planulina) wuellerstorfi for both stable and clumped isotopes analyses. Data from other benthic or infaunal species Pyrgo sp., Gyroidina soldanii, Uvigerina peregerina are only used for clumped isotopes99  with different δ13C, δ18O and Δ47 compositions and ordering states were used for monitoring and correction of the results101. Two additional reference standards (IAEA-C2 and Merck) were measured in each run to monitor the long-term reproducibility and stability of the instrument. We achieve the necessary precision by averaging ~30 clumped isotope values measured on small (70–100 μg) carbonate samples104. External reproducibility (1 standard deviation) in Δ47 of IAEA-C2 after correction was 0.033‰. The δ13C and δ18O values  of IAEA-C2 showed an external reproducibility (1 standard deviation) of 0.18‰ and 0.21‰, respectively105.Clumped isotope measurements were performed using Thermo Scientific MAT 253 and 253 Plus mass spectrometers at the GeoLab of Utrecht University. Both mass spectrometers were coupled to Thermo Fisher Scientific Kiel IV carbonate preparation devices. CO47 values (averages over ~30 separate measurements each) into temperature  using a calibration based on various recent datasets from core-top-derived foraminifera, corrected with the same carbonate standards as used in our study62:We converted the sample Δ47 -based BWTs were used in combination with δ18Obf to calculate δ18Osw (reported relative to VSMOW) with Eq. (9) of Marchitto et al.106:Δ18Obf values of the genus Cibicidoides were averaged over the same intervals as have been used for Δ47 averaging. Calibration uncertainties and measurement error were addressed by applying error propagation. The Meinicke et al.108] calibration error was propagated using the R package 109 that utilised a bootstrapped York regression slope-intercept pairs to the bootstrapped mean values for each bin. It should be noted that the calibration error is very small compared to the analytical error. The new calibration56 excludes benthic foraminifera-based data and it shifts the BWTs parallelly ~2.4 °C colder. As a result, the magnitude of cooling and relative ice volume changes are unaffected (Fig. SFor these calculations, δ111. In the standard set-up90, climate forcing follows from pre-run warm and cold snapshot climate simulations112. The applied climate forcing is transiently calculated based on the prescribed CO2 concentration and the modelled ice sheet size, through a matrix interpolation method110. Sea ice is included in the climate model forcing, but a dynamical response is not calculated by IMAU-ICE. Equilibrium experiments are performed at various CO2 levels between preindustrial and 3x preindustrial CO2 values, with insolation at present-day levels and initiated from an ice-free Miocene Antarctic topography113. Here, we perform additional sensitivity experiments, in which we apply a fixed precipitation increase and enhanced sub-shelf melt rates. The precipitation anomaly is calculated as 25% of the warm snapshot precipitation fields, sub-shelf melt rates are set to 400 m/yr115.To demonstrate the viability of a precipitation regime change leading to a fundamentally different volume-to-area ratio of the Antarctic ice sheet, we deploy the 3D thermodynamical ice sheet/shelf model IMAU-ICE v1.1.12 level (Fig. 2 level from 504 to 394 ppm and removing the anomalous forcing, leads to significantly larger ice sheet area, while the interior ice sheet height is severely reduced (Fig. These sensitivity experiments yield large-scale glaciation at a higher COvel Fig. , and an vel Fig. . Notablyced Fig.  and S7. Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1


Ice dynamic change is the primary cause of mass loss from the Antarctic Ice Sheet, thus it is important to understand the processes driving ice-ocean interactions and the timescale on which major change can occur. Here we use satellite observations to measure a rapid increase in speed and collapse of the ice shelf fronting Cadman Glacier in the absence of surface meltwater ponding. Between November 2018 and December 2019 ice speed increased by 94 ± 4% (1.47 ± 0.6 km/yr), ice discharge increased by 0.52 ± 0.21 Gt/yr, and the calving front retreated by 8 km with dynamic thinning on grounded ice of 20.1 ± 2.6 m/yr. This change was concurrent with a positive temperature anomaly in the upper ocean, where a 400 m deep channel allowed warm water to reach Cadman Glacier driving the dynamic activation, while neighbouring Funk and Lever Glaciers were protected by bathymetric sills across their fjords. Our results show that forcing by warm ocean water can cause the rapid onset of dynamic imbalance and increased ice discharge from glaciers on the Antarctic Peninsula, highlighting the region’s sensitivity to future climate variability. Warm ocean waters and favourable bathymetry caused Cadman Glacier on the Antarctic Peninsula to increase speed by 94% from 2018 to 2019. This led to increased ice discharge, glacier retreat of 8 kilometres, and glacier thinning by 20 meters per year. Similarly, surface waters in the Southern Ocean and Bellingshausen Sea on the west coast of the AP warmed by more than 1 °C from 1955 to 19942. Calving front position measurements show that ice shelves on the AP lost an area of 28,000 km2 from 1947 to 2008, with major retreat starting in the 1970s and 1980 s3. More recent records show that this trend has accelerated, with ice shelves losing an area of 20,500 km2 between 1997 and 20214, with 90% of the Peninsula’s 860 tidewater glaciers retreating in the period up to 20105. The associated loss of buttressing from ice shelves and retreat of glaciers has contributed to a substantial increase in ice mass loss from the Antarctic Peninsula Ice Sheet (APIS)9. Satellite observations show that ice loss from the region increased by 400 % between the periods 1992 to 1997 (7 ± 13 Gt/yr) and 2007 to 2012 (35 ± 17 Gt/yr), peaking in the decade after the partial collapse of the Larsen-B ice shelf in 200210. Overall, an average of 20 ± 15 Gt/yr of ice mass was lost from the APIS in the 25-years between 1992 and 201710, and ice loss is projected to increase in the future with 7 to 16 mm of sea-level equivalent by 2100 and 10 to 25 mm by 220011.Research on the Antarctic Peninsula (AP) has shown major changes in the region’s glaciers and ice shelves throughout the 20th and 21st centuries. The AP has been particularly exposed to the impact of climate change, with near surface atmospheric temperatures on the northern Peninsula warming by +0.54 °C per decade from 1951 to 201112. Since then, studies have attributed the further retreat and collapse of the Larsen-A and -B Ice Shelves on the eastern AP to thinning, foehn winds and increased summertime melt that led to hydrofracture of crevasses20. On the west AP, a pattern of increased calving front retreat on glaciers in the south has been attributed to the influence of different water masses, with cooler Bransfield Strait Water in the north and warmer Circumpolar Deep Water (CDW) in the south which is transported by the Antarctic Circumpolar Current and intrudes onto the continental shelf from the open ocean21. High temporal resolution ice speed measurements from synthetic aperture radar (SAR) satellite imagery have shown pronounced seasonal fluctuations in ice speed of up to 22.3% on west AP tidewater glaciers22 and glaciers feeding into the George VI Ice Shelf23. On the west AP, these speed fluctuations are linked with seasonal ocean warming and surface melt23, showing the sensitivity of this region to short-term variability on sub-annual timescales.Both atmospheric and oceanic forcing have driven change on the AP. In 1996, the retreat and collapse of ice shelves was linked to atmospheric warming, specifically the southerly migration of the −5 °C isotherm25. There is significant surface melt in the summertime, but less runoff compared with the east AP due to a thick, porous firn layer which takes time to saturate, and which can support perennial firn aquifers28. South of the Bransfield Strait, CDW flows onto and across the continental shelf, becoming modified CDW (mCDW) through mixing and vertical heat loss to the overlying Antarctic Surface Water layer30. This contrasts with the much colder waters of the Bransfield Strait and Weddell Sea on the east of the Peninsula31. Sea-ice on the west AP follows a seasonal pattern of summertime retreat and wintertime advance, with the wintertime sea-ice cover decreasing strongly in duration since the 1980s33. In the western Weddell Sea, a large area of sea-ice regularly persists during austral summer, becoming thicker multi-year ice. Between 1978 and 2015 sea-ice extent (SIE) in the Weddell Sea grew in extent with a small positive trend superimposed upon large interannual variability35. However, from 2016 onwards this trend reversed, with satellite data showing a reduction in SIE, reaching a record low in 2019 and contributing to record low total Antarctic SIE in 202237.The east and west sides of the AP have significantly different atmospheric and oceanographic conditions. The west AP has high accumulation due to orographically driven precipitation and strong westerly winds38. The waters of the west AP shelf support highly productive and biodiverse ecosystems which are impacted by changes in sea-ice and meltwater flux40, and glacier retreat exposes new seabed for colonisation by benthic species and enhanced carbon sequestration41. The combination of substantial long-term glaciological change, short-term seasonal variability in ice dynamics, and atmospheric and ocean forcing, have a broad impact on Antarctica’s ice and oceans and makes the ongoing study and monitoring of the AP a priority.The future evolution of glaciers on the AP has great significance for broad-scale oceanographic and glaciological changes. Model results show that increased freshwater input to the ocean from precipitation, snow, and glacier melt on the AP can strengthen the Antarctic Coastal Current, leading to enhanced basal melt rates beneath ice shelves in West Antarctica, including Pine Island and Thwaites2 from the Bruce Plateau at 2080 m elevation through a steep sided valley, which is 2 km wide at its narrowest point42. The Cadman Glacier system has four major zones inland from the fjord entrance: i) the Cadman Ice Shelf, ii) the lower glacier which we define as the fastest flowing grounded ice up to 300 m elevation, iii) the Cadman Icefall which is the steepest part of the glacier between 300 and 500 m elevation, and iv) the upper glacier which includes slower flowing ice above 500 m elevation. Satellite image records show that the calving front of Cadman Glacier was remarkably stable throughout the second half of the 20th century, with the ice-front deviating by less than 1 km from a position at the end of its fjord throughout the observational record21. The glacier catchment has a high rate of surface mass accumulation, up to 4000 mm water equivalent per year, with substantial snowmelt and runoff in the austral summer due to its location on the west coast of the AP27. Due to the snow surface conditions and the speed of ice flow, it is challenging to measure the grounding line location on this glacier using differential interferometry for which coherent Synthetic Aperture Radar (SAR) image pairs are required. Instead, the grounding line position has been measured using altimetry and photogrammetric methods43. These observations show that between 1999 and 2003 Cadman Glacier had an ice shelf confined to the lower fjord, where the grounding line was located 6 km inland of the mouth of the fjord.Cadman Glacier is located on the west coast of the AP south of Anvers Island at 65.6 °S and flows north-west into Beascochea Bay Fig. . The glaIn this study we measure ice dynamic change on Cadman Glacier over a 31-year period between 1991 and 2022. We use satellite data to measure the calving front position, ice velocity and surface elevation change, which showed that between 2018 and 2019 the glacier accelerated by 94 ± 4 % (1.47 ± 0.6 km/yr), with over 8 km of calving front retreat observed by 2021. We use direct oceanographic measurements and ocean reanalysis data to determine the environmental drivers of this major change, and contextualise it alongside the longer-term evolution of the AP.44, when the glacier terminated at the entrance to the Cadman Fjord. Prior to 2006, the calving front position did not change significantly, with variability limited to 500 m of retreat from 1985 to 1997, before it re-advanced to the 1984 position by 2001. From 2006 onwards, the glacier underwent a significant retreat of the calving front, moving inland from the fjord entrance by 1.8 km between 2005 and 2009. The glacier calving front remained in this retreated position from 2010 to 2014  grounding line location, and it remained in this position for the remainder of the study period to July 2022.Between 2014 and 2022, 6 to 12-day repeat Sentinel-1 SAR imagery resolves change in the glacier’s calving front position with much greater temporal detail than the period from 1968 to 2014. These observations show that the glacier readvanced from May 2015 to February 2018, when it reached its maximum observed extent relative to the full observational record from 1968, extending 700 m beyond the mouth of its fjord into Beascochea Bay. Although advanced, during this period the seaward 3 km of glacier tongue shows a substantial increase in damage and crevassing on the ice shelf surface and a widening of the shear margins Fig. S there is22.Our ice velocity measurements show the rate of ice flow on Cadman Glacier at high spatial and, since 2015, temporal resolution Fig. . The groOur 30-year timeseries of ice velocity Figs. b, 3b on In addition to this overall acceleration, the spatial pattern of ice velocity on Cadman Glacier changed during the study period. In 1991, there was a local speed minimum of 0.96 km/yr in the lower glacier section 700 m inland of the grounding line location Fig. . From 203, and that mass losses are entirely driven by basal melting rather than changes in the firn layer, implies a total ice shelf thinning rate of 18.5 ± 5.0 m/yr47. This result compares well with a previous study that estimated surface lowering at a rate of 2.23 m/yr between 2001 and 2010 using laser altimetry and stereo image DEMs, albeit this study included all floating and grounded ice below 1 km elevation48. Nonetheless, these results show a consistent trend in long-term thinning of Cadman Glacier’s floating shelf from 2001 until its collapse in 2021.Surface elevation measurements from CryoSat-2 were used to assess thinning on the Cadman Ice Shelf from 21st November 2010 to 10th March 2019. Between November 2010 and March 2019 prior to the ice speed acceleration, we observed a total of −16.2 ± 4.3 m surface elevation change in a sample region covering the floating ice shelf Figs. , 4a, an 49. By 2010, the local ice speed minimum was replaced by a region of constant ice speed and the 2015 REMA DEM shows that the point (iii) surface bump has disappeared. Additionally, taking the break in surface slope in the REMA DEM as an approximation for the location of Cadman Glacier’s grounding line in 2015, we find that in 2015 the grounding line is 1.25 km inland of the ASAID 1999-2003 position  shows the spatial pattern of surface elevation change on Cadman Glacier prior to the 2018/19 ice speed acceleration event. The elevation profile Fig.  shows thion Fig. . These cFurther inland, the DEMs show another surface slope inflection point 2.5 km up-glacier of the ASAID grounding line, called point (ii) Fig.  and is sSince Cadman Glacier’s rapid acceleration in 2018/19, there have also been substantial changes in the surface elevation on the inland grounded section of the glacier. Repeat track laser altimetry data from ICESat-2 between 21st April 2019 and 17th August 2021, acquired in the period immediately following the 2018/19 ice speed acceleration event show a maximum surface elevation change rate of −26.4 ± 5.1 m/yr at the base of the Cadman Icefall Fig. , and a m50. These measurements show that the main trunk of the glacier thinned by a maximum of 67.1 ± 1.5 m during this 3.3-year period, with the majority of ice volume loss occurring within a region extending 8 km upstream of the grounding line . Cadman Glacier’s location at 65.6 °S places it in a region of the AP where warm ocean waters and intrusions of CDW have been linked to glacier retreat019 Fig. . This, cLong-term thinning on the Cadman Ice Shelf can cause ice speed up through the reduction in buttressing support, likely caused by ice ungrounding from bed topographic features and a reduction in lateral resistive stresses as ice at the grounding zone thinned to the point of floatation. Although no updated direct measurement grounding line data exists for our study period, our surface elevation, elevation change, and ice velocity observations support the hypothesis that warm ocean temperatures have caused increased melting and thinning of the glacier’s ice shelf throughout the 31-year study period. Between 1991 and 2010 there was a change from a local ice speed minimum feature to an area of spatially constant ice speed immediately behind the ASAID grounding line location, which was matched by the loss of an ice surface bump between the Cook AP (2000–2009) and the REMA DEMs (2015), together with the inland migration of the break in surface slope. We interpret these changes to be indicators of a loss of basal traction and the retreat of the grounding line, from the ASAID grounding line location to a position 1.25 km further inland located near the point (ii) 2015 surface bump and associated speed minimum  from November 2018 to December 2019 and the floating ice shelf disintegrated entirely in March 2021. Surface velocity features again provide an explanation for this acceleration, as a local ice speed minimum which we associate with grounding on a bed ridge, disappeared during the period of maximum acceleration between 2018 and 2019. We interpret this as the loss of basal traction from a bed ridge and therefore conclude that thinning of the Cadman Ice Shelf and melting at the grounding zone caused the glacier to reach floatation in this section and unground from the 2015 ridge (point ii), leading to rapid grounding line retreat across a retrograde bed slope and acceleration due to the loss of basal drag and resistive stress. The Cadman Ice Shelf fully collapsed in March 2021 due to this thinning, possibly intensified through channelised melting, and the loss of pinning on the 2015 ridge. The absence of surface melt ponding on the Cadman Ice Shelf prior to its collapse demonstrates that surface ponding is not a necessary precondition for ice shelf collapse in the Antarctic Peninsula, which can occur through ocean-driven thinning and unpinning alone, similar to small ice shelf failures observed in GreenlandSince Cadman Glacier’s 2018/2019 acceleration event, the glacier has been in a state of substantial dynamic imbalance. Our ICESat-2 measurements show that since peak ice speed was reached in 2019, there has been a surface elevation change of −20.1 ± 2.6 m/yr on Cadman’s fast flowing lower glacier Fig. . No equi54. This provides evidence that decadal variability in the Southern Ocean influences ice dynamics around the Antarctic coastline as far as the west Antarctic Peninsula, even on smaller tidewater glaciers54.Our results suggest that the synchronous timing of the warm ocean water forcing can explain the rapid ice dynamic change, ice shelf collapse, and subsequent dynamic imbalance observed on Cadman Glacier. Ocean temperature from reanalysis output shows that from 2013 onwards a positive temperature anomaly was present at all depths accessible to melt the ice shelf and grounding zone Fig.  and loca56. Our results suggest differences in bathymetric structure played a strong role in the different response of Cadman and neighbouring glaciers to warming shelf water, however in future Lever and Funk Glaciers may become substantially more sensitive to ocean forcing if temperature anomalies are greater and they progressively thin in an evolution which is similar to Cadman Glacier’s but delayed. This is consistent with studies in Greenland, where bathymetric sills are important controls on ocean-fjord exchange and basal melt rates, such that the divergent behaviour of neighbouring glaciers can be explained by differences in fjord bathymetry59. Therefore, the future evolution of glaciers on the west AP coastline will likely depend strongly on the bathymetric configuration of individual embayments, adding complexity to the challenge of making accurate predictions of future mass loss.The sensitivity of Cadman Glacier, but not neighbouring Funk and Lever Glaciers, to these ocean temperature fluctuations supports our conclusion that basal melting and ungrounding of Cadman Glacier is responsible for the 2018/19 acceleration. The entrances to the Lever and Funk fjords have bathymetric sills at depths of 200 m and 230 m respectively, which limit the access of deep warm water to these glaciers compared with Cadman, so they are likely to be less sensitive to ocean forcing than Cadman Glacier which does not have a sill higher than the 350 m mid bay ridge. Sills are an effective mechanism to restrict deep, warm oceanic waters to marine-terminating glaciers, and modelling work shows that sills deeper than warm water layers can still limit basal melt rates where a hydraulically controlled circulation regime dominates60. Long-term, decadal thinning of the ice shelf and melting at the grounding zone conditioned the susceptibility of the glacier to reach floatation from its bed ridge, placing it close to the bifurcation point between advanced and retreated states. This tipping point was reached in 2018, most likely caused by the kick of an intense episode of warm water incursions onto the west AP shelf which caused the ungrounding, rapid acceleration and retreat. Our results demonstrate that such tipping points can increase ice discharge extremely rapidly, in Cadman Glacier’s case by 28.1% in 13 months. Other glaciers on the Antarctic Peninsula may be vulnerable to similar sudden acceleration and retreat, where bed conditions and bathymetry allow, similar to heterogeneous patterns of tidewater glacier retreat controlled by bed topography observed in Greenland62. For example, the largest glacier on the west coast, Fleming Glacier, has a floating tongue and is grounded on a retrograde bed slope, similar to Cadman Glacier63. Despite the relatively small size of the Antarctic Peninsula Ice Sheet compared to the West and East Antarctic Ice Sheets, the stability of the AP’s glaciers is important to global sea-level. The region has contributed 1.4 ± 0.37 mm to global mean sea-level from 1992 to 2017, 19% of the Antarctic total10. Additionally, increased freshwater input from ice discharge and newly exposed seabed due to glacier retreat would have regional-scale consequences for ocean circulation, productivity, carbon sequestration, and the ecosystem41.The rapid dynamic activation of Cadman Glacier in 2018/19 can be understood conceptually as an example of a glaciological tipping point, where an over-deepened bed geometry creates a bifurcation of steady statesOverall, the acceleration, retreat and dynamic thinning observed on Cadman Glacier since 2018 shows that glaciers on the Antarctic Peninsula can become imbalanced and increase their ice discharge into the ocean rapidly in response to forcing by warm ocean waters, highlighting the regions sensitivity to future climate variability. Our results reveal the speed and magnitude at which such glacier responses can occur and show that bed geometry and local bathymetric features are important controls on the vulnerability of glaciers in the region to such acceleration and retreat, however these glaciers are often poorly observed in terms of near ice front bathymetry and radar surveys of bed topography.This study represents a uniquely detailed assembly of data allowing us to observe and characterise the forced transition of an Antarctic glacier from one with a small floating shelf to a tidewater glacier. It highlights the impact of continuous, high-resolution multi-decadal climate observations and remote sensing campaigns which have contributed to our understanding of the complex interaction of Earth-system processes and how Antarctica will respond to a warmer climate. This work also shows the importance of continuing and improving these observations. Dense time-series of ice velocity measurements, such as those from Sentinel-1, could be further enhanced by dedicated campaigns of in-situ validation to quantify the impact of surface processes on velocity measurement error, allowing greater sensitivity to detect short-term ice dynamic changes. Substantial further insights would also be gained from frequent grounding line position measurements, particularly in regions such as the AP where low SAR coherence currently makes these measurements difficult. Improving the temporal and spatial resolution of altimetry measurements will improve the monitoring of ice shelf thinning across the Antarctic continent. Expanded airborne ground penetrating radar campaigns to measure ice thickness and bed topography near the grounding zone would reduce uncertainty in ice discharge and better characterise which glaciers are vulnerable to dynamic tipping points like the one observed in this study. Furthermore, Cadman Glacier and the Beascochea Bay system including Funk and Lever Glaciers are strong candidates for further studies and fieldwork measurements. New and sustained oceanographic measurements of the bay and individual glacier fjords behind the bathymetric sills would be valuable to understand heat transport across the west AP shelf and to compare circulation between Greenlandic fjords and the AP helping to better understand the future evolution of the Antarctic Peninsula’s glaciers.64 and precise orbit determination (POD) data from the European Space Agency. We tracked the displacement of surface features using the frequency domain intensity cross-correlation technique66 and converted this to ice velocity using the REMA 200 m DEM assuming surface parallel flow. The results were posted on a 100 m grid, and a spatially variable ice velocity error estimate was calculated by multiplying the signal to noise ratio of the cross-correlation with the ice speed67.We measured ice velocity on Cadman Glacier Fig.  by using68 and a Bayesian recursive smoother22 to compute the maximum likelihood estimate for ice speed and associated uncertainty at daily timesteps for the profile. In addition, we processed historical ice speed measurements using the same feature tracking technique applied to a pair of Landsat-5 images from 9th February 1991 and 25th February 1991, and a triplet of TerraSAR-X images from 10th November 2010 to 2nd December 2010. Optical feature tracking of Landsat images was performed with the ImGRAFT toolbox69.To assess change in Cadman Glacier’s ice speed Fig. , we extr70. Flow directions were taken from the MEaSUREs ice speed mosaic dataset71 and ice thickness was calculated from the difference between the bed elevation72 and the ice surface elevation using the REMA 100 m DEM64. We apply a surface height correction to the ice thickness data using the mean thinning rate calculated across the flux gate between 21st April 2019 and 17th August 2021, to account for the observed surface lowering during the study period  product ( ± 10 m). Change in calving front position was calculated using a curvilinear box with a 1 km width using the MaQiT tool74.We measured the change in calving front location on Cadman Glacier by manually digitizing the calving front position in Landsat-5, Landsat-7, Landsat-8 and Sentinel-2 optical imagery, and Sentinel-1 SAR imagery using the GEEDiT digitization tool75. Other remote sensing methods which rely on detecting tidal motion such as repeat track laser altimetry77 and differential range offset tracking78 do not adequately resolve Cadman Glacier. For example ICESat-2 tracks are widely spaced at this latitude and transverse to Cadman Glacier’s flow direction, so do not cover the grounding zone. To locate the grounding line position in 2015, we measure the break in surface slope of the REMA 100 m Antarctic Peninsula DEM64 between the ice shelf and grounded glacier79. We resample the REMA DEM to 10 m resolution using a cubic spline and delineate the grounding line at the most seaward local peak in surface slope gradient along the glacier’s central flowline and extend this perpendicular to flow to the shear margins.Measuring the grounding line position of Cadman Glacier in the period immediately prior to its acceleration and retreat is difficult because Sentinel-1 interferometric synthetic aperture radar (InSAR) is not coherent on the glacier, for example an automated InSAR processing and grounding line delineation campaign for 2018 was not able to resolve any grounding lines in this location80 using four repeats of track 361 from 21st April 2019, 19th Jan 2020, 17th April 2021, 17th August 2021. We differenced repeat measurements along the satellite track to calculate total surface elevation change and used a linear fit with all tracks to calculate thinning rates.We used altimetry data from the ICESat-2 laser altimeter, CryoSat-2 synthetic aperture radar interferometric (SARIn) altimeter, and stereo image DEMs to evaluate change in surface height and hence thinning of Cadman Ice Shelf and grounded inland ice. For ICESat-2, we use the ATL06 grounded ice elevation product to measure thinning of grounded ice from 2018 to 202282. There is insufficient swath-mode data to form a time-series, and so we use the Cryo-TEMPO Baseline B point of closest approach land ice (POCA_LI) product, selecting retrievals which are inside a box which covers the floating ice shelf  optimised for the AP. The A-GDEM is produced from ASTER stereo image tiles spanning 2000–2009. The REMA DEM is mosaiced from a large number of DEMs derived from several stereo-imaging satellites with a mean acquisition date of 9th May 2015 and a standard deviation of 432 days. We co-registered the Cook and REMA DEMs using the demcoreg.py package85.We also performed a direct comparison between digital elevation models from different time periods to study changing surface elevation, using the Cook et al. (2012) 100 m Antarctic Peninsula DEM (Cook DEM)86 to calculate ice thinning across the Cadman Glacier between November 2016 and February 2020. Specifically, the stereo imagery used to determine the DEM strips were acquired on 10/11/2016 and 25/02/2020. These were registered to the Mosaic over relatively stable surfaces, using the demcoreg.py package85, with masks applied to remove surface water, clouds, blunders (using the binary mask provided with the REMA product); rapidly ( > 75 m/yr) flowing ice71, open ocean and floating ice87. By co-registering both DEMs to reference elevations over slow-flowing ice, and then differencing the two DEMs, we were thus able to estimate the anomalous dynamical thinning over the Cadman glacier relative to any background climatological signal.We used repeat 2 m resolution version 4.1 Reference Elevation Model of Antarctica (REMA) strips and the version 2 REMA composite Mosaic88. We define a 0.75° x 1.5° (approx. 70 km x 85 km) region of interest to evaluate changes in the properties of water masses which may access Cadman Glacier  Copernicus Marine Service global ocean eddy-resolving reanalysis model , to evaluate ocean temperature variability in the Bellingshausen Sea, between 1993 and 2020ier Fig.  and extr89. Consequently, the AHT is an effective pathway for shelf-ocean exchange and likely for delivering heat to Cadman Glacier. The bathymetry of the west AP continental shelf is a product of past glaciations, with glacially carved throughs and over-deepened basins separated by shallow moraine sills93. These features control the flow of deep, warm ocean water toward the coast and are important for the modification of water masses through mixing94. For this reason, comparing temperatures between AHT and PD is useful to inform on the shoreward propagation of warm water masses, however, this is limited by a lack of measurements in the immediate vicinity of Beascochea Bay and Cadman Glacier.We complement this ocean potential temperature reanalysis data by using direct ocean temperature and salinity measurements from CTD’s from the Palmer Long-Term Ecological Research programme (LTER) campaign cruises on the west AP shelf. This dataset provides austral summer measurements of ocean physical properties through the water column at a regular grid of hydrographic stations on the west AP shelf and in the shelf-adjacent ocean during cruises typically conducted in January and February cruises from 1993 to 2021. Stations sampled for this dataset are not fully consistent between years, therefore, given the highly localised nature of west AP shelf conditions we ensure fair comparison between years by restricting the dataset to 16 locations sampled for at least 20 of the 28 years. There are few direct oceanographic measurements in the LTER dataset close to Cadman Glacier, the nearest points that meet our sampling threshold are the Anvers-Hugo Trough (AHT) and Palmer Deep (PD), however, these locations are suitable for studying occurrences of warm water intrusion onto the shelf and onwards to Cadman Glacier. The flow of warm CDW onto the shelf is bathymetrically constrained and observations have shown evidence of preferential cross shelf transport in large bathymetric troughs6 sample points of measured and reanalysis potential temperature, comparing all points, we find a Pearson’s correlation coefficient of 0.823  throughout the water column, with the magnitude varying with depth. We find that correlation is weakest in the 0–100 m and 100–200 m ranges, 0.672 and 0.593 respectively, with a positive bias in the reanalysis potential temperature in the 100–200 m interval, below the correlation improves significantly to 0.930 for depths below 200 m and 0.968 below 300 m . Lower correlations higher in the water column may be expected because the reanalysis output is a monthly mean, while LTER CTDs are snapshots in time, so it is likely that higher frequency variability in surface waters is not well captured in the reanalysis product. Below 200 m, however, the reanalysis and measurements agree well; these depths are where the warmest waters are found and can cause enhanced glacier melt near the grounding line, therefore, we consider the reanalysis output of sufficient representativeness and appropriate for our investigation.The GLORYS reanalysis product assimilates the Coriolis Ocean Dataset for Reanalysis (CORA), which includes the World Ocean Database and at least part of the Palmer LTER dataset, so these data are not independent, and thus a formal validation is challenging. However, we compared the GLORYS reanalysis potential temperature data and measured potential temperature from the Palmer LTER profiles to confirm the reanalysis output reflects the variability of the in situ data Fig. S. We spat m Fig. S; all cor96, supplemented by a previous study which mapped the geomorphology of Beascochea Bay from data collected in 200790.We extracted sea floor topography in the Hugo-Anvers Trough and Cadman Glacier fjord using direct bathymetric measurements from British Antarctic Survey vessels and data from the International Bathymetric Chart of the Southern Ocean (IBCSO v2)Supplementary InformationPeer Review File


Compromised structure and function of the blood-brain barrier (BBB) is one of the pathological hallmarks of brain infection by HIV-1. BBB damage during HIV-1 infection has been associated with modified expression of tight junction (TJ) proteins, including occludin. Recent evidence indicated occludin as a redox-sensitive, multifunctional protein that can act as both an NADH oxidase and influence cellular metabolism through AMPK kinase. One of the newly identified functions of occludin is its involvement in regulating HIV-1 infection. Studies suggest that occludin expression levels and the rate of HIV-1 infection share a reverse, bidirectional relationship; however, the mechanisms of this relationship are unclear. In this review, we describe the pathways involved in the regulation of HIV-1 infection by occludin. We propose that occludin may serve as a potential therapeutic target to control HIV-1 infection and to improve the lives of people living with HIV-1. Human immunodeficiency virus HIV-1) infection leads to a weakened immune system, causing people living with HIV-1 (PLH) to become susceptible to other pathogens. When the immune system becomes ineffective, an individual may develop acquired immunodeficiency syndrome (AIDS). In 2022, ~ 39 million people were living with HIV-1 and approximately 630,000 individuals had AIDS-related deaths. Since the start of the HIV-1 epidemic, tens of millions of people have died from HIV-1 and AIDS-related complications . Fo. Fo28]. occludin , 102. Anoccludin , 114. Inoccludin . In contoccludin . An assooccludin .338 has been identified as a potential phosphorylation site for these kinases  of of382 anGST–ROCK . NeverthInterestingly it has been demonstrated that inhibition of Rho-ROCK played a protective role in the BBB maintenance by limiting occludin downregulation in endothelial cells after HIV-1 Tat-treatment . 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PI437 site , 151. PD complex . PIP3 ca complex –155. Mea2 (PP2A)  and at SHLPP1/2) .Active AKT is involved in regulating many downstream targets, including mTOR, p21 proteins, Bad, caspase-9, the Wnt–β-catenin pathways, a p53 inhibitor, glycogen synthase kinase (GSK-3β), mouse double minute 2 homolog (MDM2), NF-κB, FOXOs, and cyclic adenosine monophosphate responsive element binding protein 1 (CREB-1) –164. TheThe p85 subunit of PI3K is involved in several functions, such as PTEN regulation  and phosActivation of the PI3K-Akt pathway was demonstrated to induce HIV-1 transcription by activating latent HIV-1 in monocytes and T cells . In addi490 occludin residue is the downstream phosphorylation site responsible for inducing ubiquitination of this protein [490 occludin phosphorylation. Indeed, it has been shown that inhibition of VEGF blocks occludin Ser490 phosphorylation downstream of PKCβ activation [490 phosphorylation was demonstrated to be associated with mitotic entry, in which occludin facilitates the process and increases cell proliferation [Vascular endothelial growth factor (VEGF) is an angiogenic factor that was shown to induce phosphorylation and downregulation of occludin , 186. Th protein , 188. Intivation  Fig. 3)490 occluferation . Recentlferation .VEGF has been characterized to have neuroprotective effects and was present at higher levels during neurocognitive disorders , 192. Th404, Ser408, and Thr400 - in the human occludin C-terminus. Thr375 and Ser379 have also been described in Xenopus laevis as potential CK2 phosphorylation sites [The casein kinase 1 and 2 (CK1 and CK2) are serine/threonine kinases that, among other proteins, phosphorylate occludin. There may be multiple regulatory regions on occludin that are affected by CK1-ε; at the same time, the C-terminal region of occludin can inhibit CK1-ε phosphorylation . Howeveron sites –105, 197on sites . Severalon sites . While ton sites . Moreoveon sites , 199.Occludin plays a key role in maintaining the integrity and permeability of the BBB  and it hTraditionally, occludin has been considered a multifunctional TJ protein regulating endothelial and epithelial structure and function. However, occludin is ubiquitously expressed in several cells and tissues, which suggests much broader functions than those assigned to regulating the integrity of tissue barriers. In fact, although is mainly known for its role as a TJ, occludin protein is also a multifunctional protein that can influence cellular metabolism acting as a NADH oxidase.(Table 1). Importantly, the kinases involved in occludin phosphorylation can also influence HIV-1 infection . The present review describes the cross-talks between phosphorylation of occludin and regulation of HIV-1 infection; however, it is not fully understood whether these are unrelated or causative associations. Unfortunately, no preclinical study has focused on occludin as a possible target in HIV-1 infection. We propose that a better understanding of the occludin-HIV-1 infections may identify occludin as a possible target to control HIV-1 infection and improve the life of people living with HIV-1.Recently, occludin has attracted importance due to its newly discovered metabolic functions and its role in controlling HIV-1 infection. Various mechanistic pathways have been proposed to be involved in this effect, such as regulating the expression of the OAS gene family through the STAT-1 signaling pathway, or by regulating the SIRT1 activity through NAD+. Occludin functions appear to be influenced by its phosphorylation, and several signaling pathways can be involved in this process including Src, PKC, CK2, Rho-ROCK, VEGF, and PI3K-AKT-PTEN


Caenorhabditis elegansafter a deep cryopreservation at −80°C. The expression of fungus-derived AnpIBP inC. eleganssignificantly improved its recovery rate. This result provides useful information to establish a cryopreservation technique for long-term storage using IBP molecules.Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth. IBPs have also been reported to stabilize cell membranes under non-freezing conditions. The effects of IBPs help to reduce cold- and freezing-induced damage to cells and tissues in cryopreservation. Here, we examined whether certain IBPs, namely, fish NfeIBP6 and NfeIBP8 and fungal AnpIBP1a N55D (AnpIBP), improve the recovery rate of the nematode C. elegansduring exposure to −5 °C (freezing) . Ice growth inhibition reduces the damage to tissues and cells of livingC. elegansunder freezing conditions. In addition, we found that IBPs also improve the survival rate after +2 °C and +4 °C hypothermic preservations . IBPs stabilize cell-membrane lipids at such temperaturesC. elegansafter a deep cryopreservation at −80°C.Ice-binding proteins (IBPs) inhibit the growth of ice crystals by binding to themeezing) . NfeIBP6 and NfeIBP8 were isolated fromZoarces elongatus Kner (Notched-fin eelpout). AnpIBP was recently isolated fromAntarctomyces psychrotrophicus. The TH activities of NfeIBP6, NfeIBP8 and AnpIBP are approximately 0 °C (0.4 mM), 0.5 °C (0.4 mM), and 0.7 °C (0.3 mM), respectively. Our previous paper showed that the survival rates of transgenic worms expressing IBPs in neurons or intestinal cells during exposure to freezing were lower than those of worms expressing IBPs in body wall musclesVenusin body wall muscles were 6.9% and 4.8%, respectively. In contrast, the survival rates of the worms expressing NfeIBP6, NfeIBP8, or AnpIBP in body wall muscles were 16.2%, 12.7%, and 18.9%, respectively . The recovery rate of transgenic worm expressing AnpIBP in body wall muscles was significantly higher than that of wild-type animals. IBPs presumably protected the cells from cold- and freezing-induced damage. NfeIBP6, NfeIBP8, and AnpIBP differ in TH activity but did not appear to show significant differences in the recovery rates of IBP-expressing worms. Overall, not only the inhibition of ice crystal growth but also cell membrane protection are assumed to be the key factors to improve the viability ofC. elegansin the −80 °C-cryopreservation.Here, we examined whether certain IBPs, namely, fish NfeIBP6 and NfeIBP8 and fungal AnpIBP1a N55D (AnpIBP), improve the recovery rate ofIn this study, we found that IBPs expressed in living worms improve the recovery rate after cryopreservation. The mechanism of reducing cold- and freezing-induced damage to cells and tissues in cryopreservation might be related not only to the inhibition of ice crystal growth but also to cellular protection. Further investigation is needed to understand the mechanism by which IBPs achieve cryopreservation.myo-3promoter region for the specific expression of IBPs in body wall muscles. The region containing the promoter was amplified by PCR and inserted at theXbaIandApaIsites of the plasmid pPD95.79 (a kind gift from Andrew Fire). To generate the transgenic animals, the plasmid DNAs were injected into thelin-15 (n765ts)mutant at a concentration of 30−50 ng/µL with a pbLH98lin-15(+) injection marker using a standard microinjection methodEscherichia coli OP50). Well-fed worms were used for cryopreservation. We used 3-5 medium NGM plates with large numbers of fresh L1-L4 and adult animals. The NGM plates were washed two times with 1 mL of M9 buffer. The M9 suspension with worms was collected in conical tubes. This was performed on 3-5 plates per sample. The M9 suspension was distributed 1 mL to five cryovials, and then was mixed with 1 mL of freezing solutionStrains and constructs generated previouslylin-15 (n765ts). Strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).N2, and MT1642


Tumor cell survival depends on the inter-connected activation of multiple pathways. Here, we show that acute myeloid leukemia cell lines benefit from an important inter-play between mTORC1-ERK and AKT, explaining why mTOR inhibition is rather ineffective in killing those cells. In fact, ERK1/2 activation, caused by the inhibition of the mTORC1-P70S6K dependent inhibitory loop, did not allow an efficient killing of AML cell lines. However, even ERK1/2 inhibition, together with that of mTORC1, caused a partial dephosphorylation of 4EBP1, a mTORC1 target, depending on AKT activation. Notably, AKT inhibition resulted in ERK1/2 activation. Thus, the concomitant inhibition of ERK1/2 and AKT caused a stronger 4EBP1 de-phosphorylation and was required to induce a more efficient cytotoxicity.mTOR is constitutively activated in acute myeloid leukemia (AML) cells, as indicated by the phosphorylation of its substrates, 4EBP1 and P70S6K. Here, we found that quercetin (Q) and rapamycin (Rap) inhibited P70S6K phosphorylation, partially dephosphorylated 4EBP1, and activated ERK1/2 in U937 and THP1, two leukemia cell lines. ERK1/2 inhibition by U0126 induced a stronger dephosphorylation of mTORC1 substrates and activated AKT. The concomitant inhibition of ERK1/2 and AKT further dephosphorylated 4EBP1 and further increased Q- or Rap-mediated cytotoxicity, compared to the single ERK1/2 or AKT inhibition in cells undergoing Q- or Rap-treatments. Moreover, quercetin or rapamycin reduced autophagy, particularly when used in combination with the ERK1/2 inhibitor, U0126. This effect was not dependent on TFEB localization in nuclei or cytoplasm or on the transcription of different autophagy genes, but did correlate with the reduction in protein translation due to a strong eIF2α-Ser51 phosphorylation. Thus, ERK1/2, by limiting 4EBP1 de-phosphorylation and eIF2α phosphorylation, behaves as a paladin of protein synthesis. Based on these findings, the combined inhibition of mTORC1, ERK1/2, and AKT should be considered in treatment of AML. The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase, entering in the formation of two molecular complexes, namely mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTOR maintains cell homeostasis through the regulation of many cellular functions, including transcription, translation, metabolism, autophagy, cytoskeletal organization, and cell survival ,4,5. mTOThe mTOR axis is dysregulated in many cancer cells, thus representing a promising target in anti-cancer therapy. However, it has been shown that rapamycin (Rap) and its analogs , which are allosteric inhibitors of mTORC1, can cause activation of AKT. The relief of mTORC1/P70S6K-mediated feedback inhibition of RTKs or substrates, such as platelet-derived growth factor receptor (PDGFR) ,18 and iRTK, RAS, and B-RAF [RAS is estimated to be mutated with a frequency of 15–40%  (Q) or Rap.Q, a flavonoid widely distributed in plants and fruits such as apples, onions, and tea, is known for its antioxidant, anti-inflammatory, antiproliferative, chemo-preventive, and anti-carcinogenic properties ,26. The Different signaling cascades are responsible for the control of protein translation, including the PI3K/AKT/mTORC1 and ERK pathways, through 4EBP1 dephosphorylation and the phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α) at Ser51 ,38,39. BAntibody anti-SQSTM1/p62 (BD Biosciences 610832) was purchased from BD Transduction Laboratories . Antibodies purchased from Cell Signaling Technology  were anti-4EBP1 (Cat# 9452), -phospho-ERK-Thr202/Tyr204 (Cat# 4370), -ERK (Cat# 4695), -phospho-AKT-Ser473 (Cat# 4060), -AKT (Cat# 4685), -Histone-H3 (Cat# 4499), -microtubule-associated protein 1A/1B-light chain 3 (LC3I/II) (Cat# 2775), -phospho-P70S6K-Thr389 (Cat# 9234), -P70S6K (Cat# 9202), phospho-eIF2α–Ser51 (Cat# 3398), eIF2α (Cat# 9722), -Transcription Factor EB (TFEB) (Cat# 4240), -Tubulin (Cat# 2146), horseradish peroxidase (HRP)-conjugated anti-rabbit-antibodies (Cat# 7074), and HRP-conjugated anti-mouse-antibodies (Cat# 7076). Anti-phospho-4EBP1-Ser65 (SC-293124) and radioimmunoprecipitation assay buffer (RIPA) were obtained from Santa Cruz Biotechnology Inc. . Antibodies anti-β Actin (Cat# A1978), anti-Lamin B (Cat# 6216), and anti-Puromycin (clone 12D10) (Cat# MABE343), Bafilomycin A1 (Baf), bicinchoninic acid assay (BCA), bovine serum albumin (BSA), cycloheximide (CHX), fetal calf serum (FCS), Hanks’ balanced salt solution (HBSS), Ficoll–Hypaque, l-glutamine, penicillin-streptomycin, phosphate buffered saline (PBS), propidium iodide (PI), puromycin, quercetin, RNAse, RPMI-1640, U0126, and Wortmannin were obtained from Sigma–Aldrich . Rapamycin, PF-4708671, and MK2206 were obtained from Selleckchem . Reducing agent and detergent compatible (RC-DC) protein assay, sodium dodecyl sulfate (SDS)-sample buffer, protein standard, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reagents, polyvinylidene difluoride (PVDF), and nitrocellulose membranes were from Bio-Rad Laboratories . LiteAblot TURBO chemiluminescence reagents were obtained from EuroClone . Lipofectamine RNAiMAX was obtained from Life Technologies. Other reagents of the highest purity were purchased from Bio-Rad or Sigma.2. Cells were ≥93% viable, based on calculating alive trypan blue-excluding cells in terms of the percentage of all cells counted. Washed cells were resuspended in complete medium, 1 × 106/mL, transferred to multi-well plates, treated with vehicles or inhibitors, incubated for 15 min, and subsequently exposed again to vehicles or to test agents. At the end of each experiment, aliquots of gently mixed cells were taken for PI staining and cell-cycle analysis. DMSO was used to solubilize drugs, except for puromycin, which was solubilized in H2O. The final amount of DMSO in the cultures was ≤0.3% (v/v), even when more drugs were used in combination. Mother solutions of the used drugs were Rap 5 mM, Q 100 mM, U0126 20 mM, PF-4708671 10 mM, Wortmannin 10 mM, Puromycin 10 mM, Bafilomycin 10 mM, and CHX 10 mM. Vehicle testing, even in the largest concentration, did not modify any investigated parameter in comparison with control cultures.After obtaining ethic committee approval (Prot.847/19) and informed consent from all subjects involved in the study, peripheral blood mononuclear cells (PBMC) from healthy blood donors were obtained by Ficoll–Hypaque density blood centrifugation. U-937 cell line exhibiting monocyte morphology derived from patients with histiocytic lymphoma , THP1 acv/v), pelleted again, washed twice with PBS, and finally resuspended in PBS containing RNAse (20 μg/mL) and PI (0.1 μg/mL). Events in the different cell-cycle phases were gated using an EPICS XL cytofluorimeter . At least 10,000 events in each sample were acquired. The data were analyzed using “Multicycle software for DNA content and cell cycle analysis” . SubG1 events, representative of apoptotic events, were expressed as a percentage of total cell population. Membrane permeability, indicative of cell death, was evaluated by flow cytometry (EPICS XL) after cell resuspension in HBSS containing PI (20 μg/mL) at room temperature. Analysis of samples was carried out on an EPICS-XL flow cytometer  equipped with an air-cooled argon laser tuned to 488 nm, measuring the fluorescence emission (FL3) at >575 nm. A 620 nm band pass filter was used to collect specific fluorescence signal corresponding to PI. A total of 10,000 cells were scored in each sample.Flow cytometry was used to evaluate nuclear DNA fragmentation as hypodiploic (subG1) DNA events after cell fixation and PI staining . Brieflyg centrifugation at 4 °C. An aliquot of the supernatant was used for protein quantification. Each sample was diluted to contain the same protein amount. Appropriate volumes of SDS-sample buffer (v/v) were added. Cell lysates were warmed at 95 °C for 5 min and cleared by 14,000× g centrifugation in a microfuge for 15 min at 4 °C. Equal protein amounts were separated from different samples by SDS–PAGE and transferred onto PVDF membranes. Red Ponceau staining was used to check transfer efficiency. Blots were blocked in Tris-buffered saline, Tween-20 0.1% (v/v) (TBS-T), containing BSA 5% (w/v); they were probed with specific primary antibodies, washed with TBS-T, and incubated with HRP-conjugated antibodies. For each antibody, the dilutions and incubation times suggested by the manufacturer were used. To control protein loading, the product of a housekeeping gene was probed in each membrane. Membranes with cytosol and nuclear proteins were probed with antibodies anti β-Actin, anti-Histone H3, and anti Lamin B. Immunodetection was performed using LiteAblot chemiluminescent reagents. Densitometry quantitation of bands was performed by a Mac OS X  using ImageJ software .In order to obtain cell lysates, cells were kept on ice for 30 min, vortexed every 10 min in RIPA buffer, and supplemented with proteases and inhibitors of phosphatases; then, samples were cleared by 14,000× v/v) were added to each sample. Cell lysates were warmed at 95 °C for 5 min and cleared by 14,000× g centrifugation for 15 min at 4 °C. Equal protein amounts were separated from each sample by SDS–PAGE and blotted onto nitrocellulose membranes. Transfer efficiency was checked by red Ponceau staining. Blots were blocked with 5% (w/v) milk in TBS-T at room temperature for 1 h and incubated overnight at 4 °C with mouse anti-Puromycin, clone 12D10, diluted 1:25,000. Then, membranes were washed three times for 15 min with TBS-T and incubated for 1 h at room temperature with HRP-conjugated goat anti-mouse IgG. Puromycin-labeled proteins were detected using LiteAblot chemiluminescent reagents. At 30 min from the end of the experiments, puromycin (10 μg/mL) was added to the cells. Then, as indicated above, the cells were kept for 30 min on ice in lysis buffer, and protein extraction and quantitation were performed. Appropriate volumes of unreducing SDS-sample buffer  or with scrambled siRNA , using Lipofectamine RNAiMAX according to the guidelines of the manufacturer. RPMI 1640, 20% FCS, was added after 12 h, without the removal of the transfection medium. The cells were cultured for a further 60 h. After centrifugation, the medium was replaced with RPMI 1640, 10% FCS, and the cells were treated with Q, or not treated.2O. Amplification conditions were as follows: initial denaturation at 95 °C for 15 min, followed by 40 cycles comprising denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 30 s. Cycle threshold (Ct) values, corresponding to the PCR cycle number at which the fluorescence emission reaches a threshold above the baseline emission, was determined and the mRNA expression values were calculated using β2-Microglobulin as the endogenous control according to the comparative Ct (ΔΔCt) method. Primers were purchased from Bio-Fab research  and designed as shown in Analysis of Beclin (BECN1) and TFEB target gene mRNA levels  was performed using RT-qPCR. Total RNA was isolated by TRIzol reagent and reverse transcribed using a cDNA Reverse Transcription kit. Relative RT-qPCR was performed in a total reaction volume of 20 μL, using 2 μL of cDNA, 1 μL of primer forward, 1 μL of primer reverse, 10 μL of SYBR Green qPCR Master Mix, and 6 μL Hg for 5 min at 4 °C and lysed using the Nuclear/Cytosol Fractionation Kit from BioVision , according to the manufacturer’s instructions. Briefly, cells, washed with PBS, were lysed in the cytosol extraction buffer A containing DTT and protease inhibitors, vortexed for 15 s, and incubated on ice for 10 min. Then, cytosol extraction buffer B was added to the lysates, which were incubated on ice for 1 min and centrifugated at 16,000× g for 5 min at 4 °C. Supernatants corresponding to cytoplasmic fractions were collected, while pellets were resuspended in nuclear extraction buffer mix containing DL-Dithiothreitol (DTT) and cocktail inhibitors. Each sample was vortexed for 15 s and incubated for 10 min on ice. This last step was repeated four times every 10 min. Finally, samples containing nuclear proteins were centrifuged at 16,000× g for 10 min at 4 °C and supernatants corresponding to nuclear fraction were collected. The detection of Histone H3, β-Actin, and Lamin B was used to evaluate subcellular fractions.U937 cells were collected by centrifugation at 600× The densitometry values of the bands were used to calculate the following ratio: (investigated protein/loading protein control in the same lysate)/(investigated protein in the lysate of untreated cells/loading protein control in the lysate of untreated cells).The value under each band was obtained by calculating the ratio of the densitometry results in the shown blot, as detailed above.p-value was considered statistically not significant (NS) when p ≥ 0.05, significant when p < 0.05, and very significant when p ≤ 0.01.The results were expressed as mean ± SD of at least three independent experiments. Data were analyzed using one-way ANOVA for comparisons among groups. A mTORC1 signaling represents one of the most important pro-survival pathways in AML . HoweverNext, we examined the impact of P70S6K inhibition on ERK1/2 phosphorylation and found that such inhibition by PF-4708671  further The differences observed on ERK1/2, P70S6K, and 4EBP1 occurred at the phosphorylation level, as the total amounts of these proteins were not affected by these treatments A–D. These data indicate that mTORC1 negatively regulates MEK/ERK and, therefore, mTORC1 inhibition results in ERK1/2 activation in AML cells.We next investigated the effect of ERK1/2 on mTORC1 and found that this MAPK, retrieved from the mTORC1 dependent inhibitory effect, limited mTORC1 inhibition in Rap- and Q-treated U937 and THP1 cells. In fact, the pharmacological inhibition of ERK1/2 by U0126 , which moderately decreased the constitutive phosphorylation of P70S6K and 4EBP1, caused a stronger inhibition of these mTORC1 targets in combination with Rap or Q A,B. SimiERK1/2 and mTORC1 are central players in cancer survival. Thus, we sought to investigate their role in the cell survival of AML cells. Q or Rap induced a mild cytotoxic effect in U937 cells, as Q caused an increase of PI + cells (21 ± 2.1% vs. 9 ± 3.6%) and of subG1 events (33 ± 5.8% vs. 10 ± 3.6%) A–D. ERK1These results suggest that ERK1/2, retrieved from mTORC1 inhibition, protected mTORC1 from excessive de-phosphorylation and played a pro-survival role in AML cells. We then found that ERK1/2 inhibition by U0126 was accompanied by AKT activation A,B. The Autophagy is an important catabolic process that is constitutively activated in cancer cells, by which they degrade damaged components and maintain bioenergetic levels. However, excessive or defective autophagy may promote cancer cell death. Conditions of mTORC1 inhibition are known to activate the autophagy flux . To explAltogether, these findings suggest that Rap or Q reduced the autophagy flux, particularly in combination with ERK1/2 inhibition, despite inducing a stronger inhibition of mTORC1. TFEB is a master transcription factor that coordinates the transcription of a gene network that controls the formation of autophagosomes, autophagosome-lysosome fusion, lysosome function, and biogenesis. In the unphosphorylated state, it migrates to the nucleus and induces the expression of autophagy and lysosome genes ,50,51,52Thus, the reduced autophagy flux was not due to decrease in the transcription of the investigated autophagy genes or in Q-treated cells, in which mTORC1 was partially inhibited and ERK1/2 was activated, or in cells in which ERK1/2 was inhibited and mTORC1 substrates were more strongly inhibited.The above results suggested that in Rap- and Q-treated cells, the dephosphorylation of mTORC1 substrates inhibited protein translation, leading to a decrease in autophagy-relevant proteins and, therefore, reducing autophagy. As not only dephosphorylated 4EBP1, but also eIF2α phosphorylated at Ser51, may block protein synthesis at the translational level, we next explored the activation of eIF2α in U937 and in THP1 cells and found that U0126 caused its phosphorylation at Ser51, particularly when combined with Rap or Q A,B. ThenThe above data show that the inhibition of mTORC1, leading to a protein synthesis decrease, was limited by ERK1/2 activation, and that this MAPK acted a as paladin of protein synthesis, counteracting 4EBP1 dephosphorylation and eIF2α phosphorylation.The aberrant activation of PI3K/AKT/mTOR signaling is a common finding in cancer, in which it plays a pro-survival role. The constitutive activation of PI3K/AKT/mTOR has been reported in 50–80% of AML patients, in correlation with a decrease in overall survival ; thus, kThe first aspect investigated in this study was the evaluation of whether mTORC1 inhibition could remove the inhibitory loop on ERK1/2 in AML cell lines, as shown by Carracedo et al. with respect to other tumor types . The finThen, we investigated the influence of ERK1/2 on mTORC1 in our setting, as it is known that ERK1/2 contributes to the activation of mTORC1 ,57,58. ADrosophila body fat [In the present work, we also investigated the impact of mTORC1 and ERK1/2 inhibition on the autophagy flux, as this mechanism may contribute to cell survival. mTORC1, in the presence of sufficient nutrients and growth factors, upregulates anabolic processes, including protein, lipid, and nucleotide synthesis, while it inhibits catabolic pathways, including autophagy. U937 and THP1 cells showed basal autophagy flux in the presence of the constitutive activation of mTORC1, as indicated by the phosphorylation of mTORC1 targets, P70S6K, and 4EBP1. It seems that these cells “use” mTORC1 activity to sustain the autophagy flux. Q and Rap, although causing decreased mTORC1 activity, caused a reduction of the autophagy flux, as evidenced by the expression levels of SQSTM1/p62 and LC3-II. These findings were rather surprising—particularly those obtained with Rap, as this drug is generally used to inhibit mTORC1 and, therefore, to induce autophagy. However, it should also be considered that Rap has been previously reported to attenuate autophagy induced by 6-thioguanine in colorectal cancer cells , due to body fat  and thatHere, we also examined the compartmentalization of TFEB between the nuclei and the cytoplasm, as this is the master transcription factor regulating autophagy and the expression of lysosome genes. When phosphorylated by mTORC1 and by ERK1/2, it cannot enter into the nucleus ,50. AlthCollectively, our findings indicate that in U937 and THP1 cells, there is an inhibitory loop between mTORC1/P70S6K and ERK1/2 and that ERK1/2 keeps eIF2α phosphorylation at bay A. FollowIndeed, the inhibition of ERK by U0126 removes the inhibition of ERK on eIF2α and, in presence of Rap or Q, leads to suppression of the activation loop between ERK1/2 and mTORC1, allowing a stronger dephosphorylation of mTORC1 substrates (C). Due to the stronger phosphorylation of eIF2α and the dephosphorylation of 4EBP1, cells lose two important systems that are deputed to guarantee protein synthesis and cell growth. This strategy should be kept in mind when anti-AML treatments are designed. However, the role of AKT should also be considered, as this kinase is activated following the inhibition of mTORC1 and ERK1/2.