Regulation of Net1A subcellular localization by the small GTPase Rac1

Activation of Rho family small G proteins is thought to be a critical event in breast cancer development and metastatic progression. Rho protein activation is stimulated by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs). The neuroepithelioma transforming gene 1 (Net1) is a Rho GEF specific for the RhoA subfamily that is overexpressed in primary breast tumors and breast cancer cell lines. Net1 isoform expression is also required for migration and invasion of breast cancer cells in vitro. These data indicate that Net1 may be a critical regulator of metastatic progression in breast cancer. Net1 activity is negatively regulated by sequestration in the nucleus, and relocalization of Net1 outside the nucleus is required to stimulate RhoA activation, actin cytoskeletal reorganization, and oncogenic transformation. However, regulatory mechanisms controlling the extranuclear localization of Net1 have not been identified. In this study, we have addressed the regulation of Net1A isoform localization by Rac1. Specifically, co-expression of constitutively active Rac1 with Net1A stimulates the relocalization of Net1A from the nucleus to the plasma membrane in breast cancer cells, and results in Net1A activation. Importantly, Net1A localization is also driven by endogenous Rac1 activity. Net1A relocalizes outside the nucleus in cells spreading on collagen, and when endogenous Rac1 expression was silenced by siRNA, Net1A remained nuclear in spreading cells. These data indicate that Rac1 controls the localization of the Net1A isoform and suggests a physiological role for Net1A in breast cancer cell adhesion and motility.

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

The structural and functional relationship of the p53 tumor suppressor protein

The tumor-suppressing function of p53 can be affected in a variety of manners. Here, we describe a novel mechanism of transformation by mutant p53. Previously, it had been believed that mutant p53 molecules transform cells by oligomerizing with wild-type p53 and inactivating it. However, we demonstrated that there exists an additional mechanism of inactivation of p53 available to p53 mutants. It involves sequestration of cofactors necessary to p53, and subsequent interruption of its transactivation and tumor suppression functions. The p53 amino or carboxyl termini, known to interact with a large number of cellular factors, can affect wild-type p53 in this manner. Although they are unable to oligomerize with wild-type p53, they transform cells containing p53, and inhibit its transactivation ability. In addition, they interrupt growth suppression by p53, but not RB, confirming that they specifically affect p53 function, rather than having a general growth-stimulatory phenomenon. Also, we have cloned a p53 tumor mutation which results in expression of the amino terminus of p53. This provides a means to study the factor-sequestration transforming mechanism in vivo. Additionally, we found that the published sequence of the mdm2 gene is in error. mdm2 is a gene intimately involved with p53, blocking its ability to transform cells. Finally, previous data had established the influence of cell-cycle status on p53 function. In growth-arrested cells, wild-type p53 expressed by a transgene cannot activate transcription, but if these cells are forced to cycle by addition of cyclin E, p53 once again becomes functional. In this study, we extend these findings by examining only those cells successfully transfected, using fluorescence-activated cell sorting. Our results support the previous data, that cyclin E pushes growth-arrested cells back into the cell cycle. In summary, we have demonstrated the potential importance of cofactor association and protein modification to the abilities of p53 to cause transcription activation and repression, inhibition of DNA replication and induction of DNA repair, and initiation of cell-cycle arrest and apoptosis. Further elucidation of these processes and their roles in tumor suppression will prove fascinating indeed. ^

Dissolved organic carbon (DOC) in Arctic ground ice, from northwest Canada, east Siberia, and Alaska

Thermal permafrost degradation and coastal erosion in the Arctic remobilize substantial amounts of organic carbon (OC) and nutrients which have accumulated in late Pleistocene and Holocene unconsolidated deposits. Permafrost vulnerability to thaw subsidence, collapsing coastlines and irreversible landscape change are largely due to the presence of large amounts of massive ground ice such as ice wedges. However, ground ice has not, until now, been considered to be a source of dissolved organic carbon (DOC), dissolved inorganic carbon (DIC) and other elements which are important for ecosystems and carbon cycling. Here we show, using biogeochemical data from a large number of different ice bodies throughout the Arctic, that ice wedges have the greatest potential for DOC storage, with a maximum of 28.6 mg/L (mean: 9.6 mg/L). Variation in DOC concentration is positively correlated with and explained by the concentrations and relative amounts of typically terrestrial cations such as Mg2+ and K+. DOC sequestration into ground ice was more effective during the late Pleistocene than during the Holocene, which can be explained by rapid sediment and OC accumulation, the prevalence of more easily degradable vegetation and immediate incorporation into permafrost. We assume that pristine snowmelt is able to leach considerable amounts of well-preserved and highly bioavailable DOC as well as other elements from surface sediments, which are rapidly frozen and stored in ground ice, especially in ice wedges, even before further degradation. We found that ice wedges in the Yedoma region represent a significant DOC (45.2 Tg) and DIC (33.6 Tg) pool in permafrost areas and a freshwater reservoir of 4200 km**3. This study underlines the need to discriminate between particulate OC and DOC to assess the availability and vulnerability of the permafrost carbon pool for ecosystems and climate feedback upon mobilization.

(Table 2) Survivorship of transplanted seedlings of Picea glauca inside and outside three tree islands in 1994, 1995, 2009 and after 15 years

The northern boundary of boreal forest and the ranges of tree species are expected to shift northward in response to climate warming, which will result in a decrease in the albedo of areas currently covered by tundra vegetation, an increase in terrestrial carbon sequestration, and an alteration of biodiversity in the current Low Arctic. Central to the prediction of forest expansion is an increase in the reproductive capacity and establishment of individual trees. We assessed cone production, seed viability, and transplanted seedling success of Picea glauca (Moench.) Voss. (white spruce) in the early 1990s and again in the late 2000s at four forest stand sites and eight tree island sites (clonal populations beyond present treeline) in the Mackenzie Delta region of the Northwest Territories, Canada. Over the past 20 years, average temperatures in this region have increased by 0.9 °C. This area has the northernmost forest-tundra ecotone in North America and is one of the few circumpolar regions where the northern limit of conifer trees reaches the Arctic Ocean. We found that cone production and seed viability did not change between the two periods of examination and that both variables decreased northward across the forest-tundra ecotone. Nevertheless, white spruce individuals at the northern limit of the forest-tundra ecotone produced viable seeds. Furthermore, transplanted seedlings were able to survive in the northernmost sites for 15 years, but there were no signs of natural regeneration. These results indicate that if climatic conditions continue to ameliorate, reproductive output will likely increase, but seedling establishment and forest expansion within the forest-tundra of this region is unlikely to occur without the availability of suitable recruitment sites. Processes that affect the availability of recruitment sites are likely to be important elsewhere in the circumpolar ecotone, and should be incorporated into models and predictions of climate change and its effects on the northern forest-tundra ecotone.

Iron (Fe) concentrations and fluxes in TALDICE ice core 0.6 to 314 ky BP

Atmospheric fluxes of iron (Fe) over the past 200 kyr are reported for the coastal Antarctic Talos Dome ice core, based on acid leachable Fe concentrations. Fluxes of Fe to Talos Dome were consistently greater than those at Dome C, with the greatest difference observed during interglacial climates. We observe different Fe flux trends at Dome C and Talos Dome during the deglaciation and early Holocene, attributed to a combination of deglacial activation of dust sources local to Talos Dome and the reorganisation of atmospheric transport pathways with the retreat of the Ross Sea ice shelf. This supports similar findings based on dust particle sizes and fluxes and Rare Earth Element fluxes. We show that Ca and Fe should not be used as quantitative proxies for mineral dust, as they all demonstrate different deglacial trends at Talos Dome and Dome C. Considering that a 20 ppmv decrease in atmospheric CO2 at the coldest part of the last glacial maximum occurs contemporaneously with the period of greatest Fe and dust flux to Antarctica, we confirm that the maximum contribution of aeolian dust deposition to Southern Ocean sequestration of atmospheric CO2 is approximately 20 ppmv.

Cenomanian/Turonian sea surface temperatures of the equatorial Atlantic reconstructed from geochemical data

Oceanic anoxic event 2 (OAE-2) occurring during the Cenomanian/Turonian (C/T) transition is evident from a globally recognized positive stable carbon isotopic excursion and is thought to represent one of the most extreme carbon cycle perturbations of the last 100 Myr. However, the impact of this major perturbation on and interaction with global climate remains unclear. Here we report new high-resolution records of sea surface temperature (SST) based on TEX86 and d 18O of excellently preserved planktic foraminifera and stable organic carbon isotopes across the C/T transition from black shales located offshore Suriname/French Guiana (Demerara Rise, Ocean Drilling Program Leg 207 Site 1260) and offshore Senegal (Cape Verde Basin, Deep Sea Drilling Project Leg 41 Site 367). At Site 1260, where both SST proxy records can be determined, a good match between conservative SST estimates from TEX86 and d 18O is observed. We find that late Cenomanian SSTs in the equatorial Atlantic Ocean (33°C) were substantially warmer than today (27°-29°C) and that the onset of OAE-2 coincided with a rapid shift to an even warmer (35°-36°C) regime. Within the early stages of the OAE a marked (4°C) cooling to temperatures lower than pre-OAE conditions is observed. However, well before the termination of OAE-2 the warm regime was reestablished and persisted into the Turonian. Our findings corroborate the view that the C/T transition represents the onset of the interval of peak Cretaceous warmth. More importantly, they are consistent with the hypotheses that mid-Cretaceous warmth can be attributed to high levels of atmospheric carbon dioxide (CO2) and that major OAEs were capable of triggering global cooling through the negative feedback effect of organic carbon-burial-led CO2 sequestration. Evidently, however, the factors that gave rise to the observed shift to a warmer climate regime at the onset of OAE-2 were sufficiently powerful that they were only briefly counterbalanced by the high rates of carbon burial attained during even the most extreme interval of organic carbon burial in the last 100 Myr.

Age determination of ODP Hole 145-887

Atmospheric carbon dioxide concentrations were significantly lower during glacial periods than during intervening interglacial periods, but the mechanisms responsible for this difference remain uncertain. Many recent explanations call on greater carbon storage in a poorly ventilated deep ocean during glacial periods (Trancois et al., 1997, doi:10.1038/40073; Toggweiler, 1999, doi:10.1029/1999PA900033; Stephens and Keeling, 2000, doi:10.1038/35004556; Marchitto et al., 2007, doi:10.1126/science.1138679; Sigman and Boyle, 2000, doi:10.1038/35038000), but direct evidence regarding the ventilation and respired carbon content of the glacial deep ocean is sparse and often equivocal (Broecker et al., 2004, doi:10.1126/science.1102293). Here we present sedimentary geochemical records from sites spanning the deep subarctic Pacific that -together with previously published results (Keigwin, 1998, doi:10.1029/98PA00874)- show that a poorly ventilated water mass containing a high concentration of respired carbon dioxide occupied the North Pacific abyss during the Last Glacial Maximum. Despite an inferred increase in deep Southern Ocean ventilation during the first step of the deglaciation (18,000-15,000 years ago) (Marchitto et al., 2007, doi:10.1126/science.1138679; Monnin et al., 2001, doi:10.1126/science.291.5501.112), we find no evidence for improved ventilation in the abyssal subarctic Pacific until a rapid transition ~14,600 years ago: this change was accompanied by an acceleration of export production from the surface waters above but only a small increase in atmospheric carbon dioxide concentration (Monnin et al., 2001, doi:10.1126/science.291.5501.112). We speculate that these changes were mechanistically linked to a roughly coeval increase in deep water formation in the North Atlantic (Robinson et al., 2005, doi:10.1126/science.1114832; Skinner nd Shackleton, 2004, doi:10.1029/2003PA000983; McManus et al., 2004, doi:10.1038/nature02494), which flushed respired carbon dioxide from northern abyssal waters, but also increased the supply of nutrients to the upper ocean, leading to greater carbon dioxide sequestration at mid-depths and stalling the rise of atmospheric carbon dioxide concentrations. Our findings are qualitatively consistent with hypotheses invoking a deglacial flushing of respired carbon dioxide from an isolated, deep ocean reservoir periods (Trancois et al., 1997, doi:10.1038/40073; Toggweiler, 1999, doi:10.1029/1999PA900033; Stephens and Keeling, 2000, doi:10.1038/35004556; Marchitto et al., 2007, doi:10.1126/science.1138679; Sigman and Boyle, 2000, doi:10.1038/35038000; Boyle, 1988, doi:10.1038/331055a0), but suggest that the reservoir may have been released in stages, as vigorous deep water ventilation switched between North Atlantic and Southern Ocean source regions.

Geochemistry on Holocene sediments from the Mediterranean

The enhanced accumulation of organic matter in Eastern Mediterranean sapropels and their unusually low d15N values have been attributed to either enhanced nutrient availability which led to elevated primary production and carbon sequestration or to enhanced organic matter preservation under anoxic conditions. In order to evaluate these two hypothesis we have determined Ba/Al ratios, amino acid composition, N and organic C concentrations and d15N in sinking particles, surface sediments, eight spatially distributed core records of the youngest sapropel S1 (10-6 ka) and older sapropels (S5, S6) from two locations. These data suggest that (i) temporal and spatial variations in d15N of sedimentary N are driven by different degrees of diagenesis at different sites rather than by changes in N-sources or primary productivity and (ii) present day TOC export production would suffice to create a sapropel like S1 under conditions of deep-water anoxia. This implies that both enhanced TOC accumulation and d15N depletion in sapropels were due to the absence of oxygen in deep waters. Thus preservation plays a major role for the accumulation of organic-rich sediments casting doubt on the need of enhanced primary production for sapropel formation.

Age-depth relation and red colour reflectance of ODP Leg 177 sites

The Antarctic Polar Front is an important biogeochemical divider in the Southern Ocean. Laminated diatom mat deposits record episodes of massive flux of the diatom Thalassiothrix antarctica beneath the Antarctic Polar Front and provide a marker for tracking the migration of the Front through time. Ocean Drilling Program Sites 1091, 1093 and 1094 are the only deep piston cored record hitherto sampled from the sediments of the circumpolar biogenic opal belt. Mapping of diatom mat deposits between these sites indicates a glacial-interglacial front migration of up to 6 degrees of latitude in the early/mid Pleistocene. The mid-Pleistocene transition marks a stepwise minimum 7° northward migration of the locus of the Polar Front sustained for about 450 kyr until an abrupt southward return to a locus similar to its modern position and further south than any mid-Pleistocene locus. This interval from a "900 ka event" that saw major cooling of the oceans and a d13C minimum through to the 424 ka Mid-Brunhes Event at Termination V is also seemingly characterised by 1) sustained decreased carbonate in the sub-tropical south Atlantic, 2) reduced strength of Antarctic deep meridional circulation, 3) lower interglacial temperatures and lower interglacial atmospheric CO2 levels (by some 30 per mil) than those of the last 400 kyr, evidencing less complete deglaciation. This evidence is consistent with a prolonged period lasting 450 kyr of only partial ventilation of the deep ocean during interglacials and suggests that the mechanisms highlighted by recent hypotheses linking mid-latitude atmospheric conditions to the extent of deep ocean ventilation and carbon sequestration over glacial-interglacial cycles are likely in operation during the longer time scale characteristic of the mid-Pleistocene transition. The cooling that initiated the "900 ka event" may have been driven by minima in insolation amplitude related to eccentricity modulation of precession that also affected low latitude climates as marked by threshold changes in the African monsoon system. The major thresholds in earth system behaviour through the mid-Pleistocene transition were likely governed by an interplay of the 100 kyr and 400 kyr eccentricity modulation of precession.

Diatom abundances of ODP Hole 177-1093A in the Southern Ocean

Laminated diatom ooze samples collected during ODP Leg 177 were analysed using scanning electron microscope (SEM) and optical microscopy to test their potential as high-resolution records of Polar Front hydrography, surface production, and export. SEM analysis from two intervals, marine isotope stage (MIS) 29 and 12/11, respectively, recovered from 50°S in the Atlantic Ocean (ODP Site 1093, Hole A, sections 13H-4 0-18 cm and 23H-4 0-22 cm), show abundant and well-preserved Thalassiothrix antarctica mats, thought to be indicative of rapid export from the surface and deposition in the sediment. A preliminary analysis of laminae succession points to a possible annual couplet/triplet succession of laminae, and suggests exceptionally high local sedimentation rates of 57 and 80 cm/kyr for MIS 12/11 and 29, respectively. Such high accumulation rates imply that local export from the surface layer and sequestration of biogenic silica and organic matter to the sediments may have been much higher than previously suggested.

Phosphorus components and barite in untreated and in carbonate-free sediments of ODP Hole 199-1221C

We determined changes in equatorial Pacific phosphorus (µmol P/g) and barite (BaSO4; wt%) concentrations at high resolution (2 cm) across the Paleocene/Eocene (P/E) boundary in sediments from Ocean Drilling Program (ODP) Leg 199 Site 1221 (153.40 to 154.80 meters below seafloor [mbsf]). Oxide-associated, authigenic, and organic P sequentially extracted from bulk sediment were used to distinguish reactive P from detrital P. We separated barite from bulk sediment and compared its morphology with that of modern unaltered biogenic barite to check for diagenesis. On a CaCO3-free basis, reactive P concentrations are relatively constant and high (323 µmol P/g or ~1 wt%). Barite concentrations range from 0.05 to 5.6 wt%, calculated on a CaCO3-free basis, and show significant variability over this time interval. Shipboard measurements of P and Ba in bulk sediments are systematically lower (by ~25%) than shore-based concentrations and likely indicate problems with shipboard standard calibrations. The presence of Mn oxides and the size, crystal morphology, and sulfur isotopes of barite imply deposition in sulfate-rich pore fluids. Relatively constant reactive P, organic C, and biogenic silica concentrations calculated on a CaCO3-free basis indicate generally little variation in organic C, reactive P, and biogenic opal burial across the P/E boundary, whereas variable barite concentrations indicate significant changes in export productivity. Low barite Ba/reactive P ratios before and immediately after the Benthic Extinction Event (BEE) may indicate efficient nutrient burial, and, if nutrient burial and organic C burial are linked, high relative organic C burial that could temporarily drawdown CO2 at this site. This interpretation requires postdepositional oxidation of organic C because organic C to reactive P ratios are low throughout the section. After the BEE, higher barite Ba/reactive P ratios combined with higher barite Ba concentrations may imply that higher export productivity was coupled with unchanged reactive P burial, indicating efficient nutrient and possibly also organic C recycling in the water column. If the nutrient recycling is decoupled from organic C, the high export production could be indicative of drawdown of CO2. However, the observation that organic C burial is not high where barite burial is high may imply that either C sequestration was restricted to the deep ocean and thus occurred only on timescales of the deep ocean mixing or that postdepositional oxidation (burn down) of organic matter affected the sediments. The decoupling of barite and opal may result from low opal preservation or production that is not diatom based.

Geochemistry of Baltic Sea sediments

The Baltic Sea has experienced three major intervals of bottom water hypoxia following the intrusion of seawater ca. 8 kyrs ago. These intervals occurred during the Holocene Thermal Maximum (HTM), Medieval Climate Anomaly (MCA) and during recent decades. Here, we show that sequestration of both Fe and Mn in Baltic Sea sediments generally increases with water depth, and we attribute this to shelf-to-basin transfer ("shuttling") of Fe and Mn. Burial of Mn in slope and basin sediments was enhanced following the lake-brackish/marine transition at the beginning of the hypoxic interval during the HTM. During hypoxic intervals, shelf-to-basin transfer of Fe was generally enhanced but that of Mn was reduced. However, intensification of hypoxia within hypoxic intervals led to decreased burial of both Mn and Fe in deep basin sediments. This implies a non-linearity in shelf Fe release upon expanding hypoxia with initial enhanced Fe release relative to oxic conditions followed by increased retention in shelf sediments, likely in the form of iron sulfide minerals. For Mn, extended hypoxia leads to more limited sequestration as Mn carbonate in deep basin sediments, presumably because of more rapid reduction of Mn oxides formed after inflows and subsequent escape of dissolved Mn to the overlying water. Our Fe records suggest that modern Baltic Sea hypoxia is more widespread than in the past. Furthermore, hypoxia-driven variations in shelf-to-basin transfer of Fe may have impacted the dynamics of P and sulfide in the Baltic Sea thus providing potential feedbacks on the further development of hypoxia.

Stable isotope record and sediment composition of the subantarctic Pacific

Benthic foraminiferal stable carbon isotope records from the South Atlantic show significant declines toward more "Pacific-like" values at ~7 and ~2.7 Ma, and it has been posited that these shifts may mark steps toward increased CO2 sequestration in the deep Southern Ocean as climate cooled over the late Neogene. We generated new stable isotope records from abyssal subantarctic Pacific cores MV0502-4JC and ELT 25-11. The record from MV0502-4JC suggests that the Southern Ocean remained well mixed and free of vertical or interbasinal d13C gradients following the late Miocene carbon shift (LMCS). According to the records from MV0502-4JC and ELT 25-11, however, cold, low d13C bottom waters developed in the Southern Ocean in the late Pliocene and persisted until ~1.7 Ma. These new data suggest that while conditions in the abyssal Southern Ocean following the LMCS were comparable to the present day, sequestration of respired CO2 may have increased in the deepest parts of the Southern Ocean during the late Pliocene, a critical period for the growth and establishment of the Northern Hemisphere ice sheets.

(Tabls S1) Bulk geochemistry for sediment samples collected from the Cenomanian-Turonian boundary section at Pont d'Issole

Oceanic Anoxic Event 2 (OAE2), spanning the Cenomanian-Turonian boundary (CTB), represents one of the largest perturbations in the global carbon cycle in the last 100 Myr. The d13Ccarb, d13Corg, and d18O chemostratigraphy of a black shale-bearing CTB succession in the Vocontian Basin of France is described and correlated at high resolution to the European CTB reference section at Eastbourne, England, and to successions in Germany, the equatorial and midlatitude proto-North Atlantic, and the U.S. Western Interior Seaway (WIS). Delta13C (offset between d13Ccarb and d13Corg) is shown to be a good pCO2 proxy that is consistent with pCO2 records obtained using biomarker d13C data from Atlantic black shales and leaf stomata data from WIS sections. Boreal chalk d18O records show sea surface temperature (SST) changes that closely follow the Delta13C pCO2 proxy and confirm TEX86 results from deep ocean sites. Rising pCO2 and SST during the Late Cenomanian is attributed to volcanic degassing; pCO2 and SST maxima occurred at the onset of black shale deposition, followed by falling pCO2 and cooling due to carbon sequestration by marine organic productivity and preservation, and increased silicate weathering. A marked pCO2 minimum (~25% fall) occurred with a SST minimum (Plenus Cold Event) showing >4°C of cooling in ~40 kyr. Renewed increases in pCO2, SST, and d13C during latest Cenomanian black shale deposition suggest that a continuing volcanogenic CO2 flux overrode further drawdown effects. Maximum pCO2 and SST followed the end of OAE2, associated with a falling nutrient supply during the Early Turonian eustatic highstand.