Radiocarbon constraints on the glacial ocean circulation and its impact on atmospheric CO2

While the ocean’s large-scale overturning circulation is thought to have been significantly different under the climatic conditions of the Last Glacial Maximum (LGM), the exact nature of the glacial circulation and its implications for global carbon cycling continue to be debated. Here we use a global array of ocean–atmosphere radiocarbon disequilibrium estimates to demonstrate a ∼689±53 14C-yr increase in the average residence time of carbon in the deep ocean at the LGM. A predominantly southern-sourced abyssal overturning limb that was more isolated from its shallower northern counterparts is interpreted to have extended from the Southern Ocean, producing a widespread radiocarbon age maximum at mid-depths and depriving the deep ocean of a fast escape route for accumulating respired carbon. While the exact magnitude of the resulting carbon cycle impacts remains to be confirmed, the radiocarbon data suggest an increase in the efficiency of the biological carbon pump that could have accounted for as much as half of the glacial–interglacial CO2 change

Radiocarbon constraints on the glacial ocean circulation and its impact on atmospheric CO2_2

While the ocean's large-scale overturning circulation is thought to have been significantly different under the climatic conditions of the Last Glacial Maximum (LGM), the exact nature of the glacial circulation and its implications for global carbon cycling continue to be debated. Here we use a global array of ocean-atmosphere radiocarbon disequilibrium estimates to demonstrate a ~689±53 $^{14}$C-yr increase in the average residence time of carbon in the deep ocean at the LGM. A predominantly southern-sourced abyssal overturning limb that was more isolated from its shallower northern counterparts is interpreted to have extended from the Southern Ocean, producing a widespread radiocarbon age maximum at mid-depths and depriving the deep ocean of a fast escape route for accumulating respired carbon. While the exact magnitude of the resulting carbon cycle impacts remains to be confirmed, the radiocarbon data suggest an increase in the efficiency of the biological carbon pump that could have accounted for as much as half of the glacial-interglacial CO$_2$ change.This work was made possible by NERC grant NE/L006421/1, and was supported by NERC radiocarbon analysis allocation number 1245.1007, as well as the Royal Society and the Cambridge Isaac Newton Trust

epsilon-Polylysine-Capped Mesoporous Silica Nanoparticles as Carrier of the C9h Peptide to Induce Apoptosis in Cancer Cells

[EN] Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Ac alpha interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Ac alpha interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safraninO (S2) or with C9h peptide (S4) and functionalized with epsilon-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safraninO or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50%) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Ac alpha interaction.The authors wish to express their gratitude to the Spanish government (Projects MAT2015-64139-C4-1, SAF2012-31405, SAF2015-67077-R, AGL2015-70235-C2-2-R (MINECO/FEDER)), the Generalitat Valencia (Projects PROMETEOII/2014/047, PROMETEO/2012/061) and the CIBER-BBN for their support. C.T. is grateful to the Spanish Ministry of Science and Innovation for her Ph.D. fellowship.De La Torre-Paredes, C.; Domínguez-Berrocal, L.; Murguía, JR.; Marcos Martínez, MD.; Martínez-Máñez, R.; Bravo, J.; Sancenón Galarza, F. (2018). epsilon-Polylysine-Capped Mesoporous Silica Nanoparticles as Carrier of the C9h Peptide to Induce Apoptosis in Cancer Cells. Chemistry - A European Journal. 24(8):1890-1897. https://doi.org/10.1002/chem.201704161S18901897248Lyon, M. A., Ducruet, A. P., Wipf, P., & Lazo, J. S. (2002). Dual-specificity phosphatases as targets for antineoplastic agents. Nature Reviews Drug Discovery, 1(12), 961-976. doi:10.1038/nrd963Ducret, F., Turc-Baron, C., Pointet, P., Vernin, G., Skowron, O., Mc Gregor, B., … Vincent, M. (2005). Tumeur à rénine. À propos d’un nouveau cas diagnostiqué au cours d’une grossesse. Néphrologie & Thérapeutique, 1(1), 52-61. doi:10.1016/j.nephro.2005.01.008Lazar, D. F., & Saltiel, A. R. (2006). Lipid phosphatases as drug discovery targets for type 2 diabetes. Nature Reviews Drug Discovery, 5(4), 333-342. doi:10.1038/nrd2007Tonks, N. K. (2006). Protein tyrosine phosphatases: from genes, to function, to disease. 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International Journal of Cancer, 65(2), 230-237. doi:10.1002/(sici)1097-0215(19960117)65:23.0.co;2-hLiang, X.-J., Chen, C., Zhao, Y., & Wang, P. C. (2009). Circumventing Tumor Resistance to Chemotherapy by Nanotechnology. Multi-Drug Resistance in Cancer, 467-488. doi:10.1007/978-1-60761-416-6_21He, Q., & Shi, J. (2013). MSN Anti-Cancer Nanomedicines: Chemotherapy Enhancement, Overcoming of Drug Resistance, and Metastasis Inhibition. Advanced Materials, 26(3), 391-411. doi:10.1002/adma.201303123Ding, C., & Li, Z. (2017). A review of drug release mechanisms from nanocarrier systems. Materials Science and Engineering: C, 76, 1440-1453. doi:10.1016/j.msec.2017.03.130Llopis-Lorente, A., Lozano-Torres, B., Bernardos, A., Martínez-Máñez, R., & Sancenón, F. (2017). Mesoporous silica materials for controlled delivery based on enzymes. Journal of Materials Chemistry B, 5(17), 3069-3083. doi:10.1039/c7tb00348jTibbitt, M. W., Dahlman, J. E., & Langer, R. (2016). Emerging Frontiers in Drug Delivery. Journal of the American Chemical Society, 138(3), 704-717. doi:10.1021/jacs.5b09974Mai, W. X., & Meng, H. (2012). Mesoporous silica nanoparticles: A multifunctional nano therapeutic system. Integrative Biology, 5(1), 19-28. doi:10.1039/c2ib20137bDoadrio, A., Salinas, A., Sánchez-Montero, J., & Vallet-Regí, M. (2015). Drug release from ordered mesoporous silicas. Current Pharmaceutical Design, 21(42), 6213-6819. doi:10.2174/1381612822666151106121419Argyo, C., Weiss, V., Bräuchle, C., & Bein, T. (2013). Multifunctional Mesoporous Silica Nanoparticles as a Universal Platform for Drug Delivery. Chemistry of Materials, 26(1), 435-451. doi:10.1021/cm402592tGagliardi, M. (2017). Recent Advances in Preclinical Studies and Potential Applications of Dendrimers as Drug Carriers in the Central Nervous System. Current Pharmaceutical Design, 23(21). doi:10.2174/1381612823666170313124811Zhang, R. X., Ahmed, T., Li, L. Y., Li, J., Abbasi, A. Z., & Wu, X. Y. (2017). Design of nanocarriers for nanoscale drug delivery to enhance cancer treatment using hybrid polymer and lipid building blocks. Nanoscale, 9(4), 1334-1355. doi:10.1039/c6nr08486aGuo, X., Wang, L., Wei, X., & Zhou, S. (2016). Polymer-based drug delivery systems for cancer treatment. Journal of Polymer Science Part A: Polymer Chemistry, 54(22), 3525-3550. doi:10.1002/pola.28252Li, Z., Barnes, J. C., Bosoy, A., Stoddart, J. F., & Zink, J. I. (2012). Mesoporous silica nanoparticles in biomedical applications. Chemical Society Reviews, 41(7), 2590. doi:10.1039/c1cs15246gWang, Y., Zhao, Q., Han, N., Bai, L., Li, J., Liu, J., … Wang, S. (2015). Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomedicine: Nanotechnology, Biology and Medicine, 11(2), 313-327. doi:10.1016/j.nano.2014.09.014Sun, R., Wang, W., Wen, Y., & Zhang, X. (2015). Recent Advance on Mesoporous Silica Nanoparticles-Based Controlled Release System: Intelligent Switches Open up New Horizon. Nanomaterials, 5(4), 2019-2053. doi:10.3390/nano5042019Mura, S., Nicolas, J., & Couvreur, P. (2013). Stimuli-responsive nanocarriers for drug delivery. Nature Materials, 12(11), 991-1003. doi:10.1038/nmat3776Tarn, D., Ashley, C. E., Xue, M., Carnes, E. C., Zink, J. I., & Brinker, C. J. (2013). Mesoporous Silica Nanoparticle Nanocarriers: Biofunctionality and Biocompatibility. Accounts of Chemical Research, 46(3), 792-801. doi:10.1021/ar3000986Aznar, E., Oroval, M., Pascual, L., Murguía, J. R., Martínez-Máñez, R., & Sancenón, F. (2016). Gated Materials for On-Command Release of Guest Molecules. Chemical Reviews, 116(2), 561-718. doi:10.1021/acs.chemrev.5b00456Agostini, A., Mondragón, L., Bernardos, A., Martínez-Máñez, R., Marcos, M. D., Sancenón, F., … Murguía, J. R. (2012). Targeted Cargo Delivery in Senescent Cells Using Capped Mesoporous Silica Nanoparticles. Angewandte Chemie International Edition, 51(42), 10556-10560. doi:10.1002/anie.201204663Agostini, A., Mondragón, L., Bernardos, A., Martínez-Máñez, R., Marcos, M. D., Sancenón, F., … Murguía, J. R. (2012). Targeted Cargo Delivery in Senescent Cells Using Capped Mesoporous Silica Nanoparticles. Angewandte Chemie, 124(42), 10708-10712. doi:10.1002/ange.201204663Agostini, A., Mondragón, L., Coll, C., Aznar, E., Marcos, M. D., Martínez-Máñez, R., … Amorós, P. (2012). Dual Enzyme-Triggered Controlled Release on Capped Nanometric Silica Mesoporous Supports. ChemistryOpen, 1(1), 17-20. doi:10.1002/open.201200003Aznar, E., Villalonga, R., Giménez, C., Sancenón, F., Marcos, M. D., Martínez-Máñez, R., … Amorós, P. (2013). Glucose-triggered release using enzyme-gated mesoporous silica nanoparticles. Chemical Communications, 49(57), 6391. doi:10.1039/c3cc42210kGiménez, C., de la Torre, C., Gorbe, M., Aznar, E., Sancenón, F., Murguía, J. R., … Amorós, P. (2015). Gated Mesoporous Silica Nanoparticles for the Controlled Delivery of Drugs in Cancer Cells. Langmuir, 31(12), 3753-3762. doi:10.1021/acs.langmuir.5b00139De la Torre, C., Casanova, I., Acosta, G., Coll, C., Moreno, M. J., Albericio, F., … Martínez-Máñez, R. (2014). Gated Mesoporous Silica Nanoparticles Using a Double-Role Circular Peptide for the Controlled and Target-Preferential Release of Doxorubicin in CXCR4-Expresing Lymphoma Cells. Advanced Functional Materials, 25(5), 687-695. doi:10.1002/adfm.201403822De la Torre, C., Agostini, A., Mondragón, L., Orzáez, M., Sancenón, F., Martínez-Máñez, R., … Pérez-Payá, E. (2014). Temperature-controlled release by changes in the secondary structure of peptides anchored onto mesoporous silica supports. Chem. Commun., 50(24), 3184-3186. doi:10.1039/c3cc49421gAznar, E., Coll, C., Marcos, M. D., Martínez-Máñez, R., Sancenón, F., Soto, J., … Ruiz, E. (2009). Borate-Driven Gatelike Scaffolding Using Mesoporous Materials Functionalised with Saccharides. Chemistry - A European Journal, 15(28), 6877-6888. doi:10.1002/chem.200900090Bringas, E., Köysüren, Ö., Quach, D. V., Mahmoudi, M., Aznar, E., Roehling, J. D., … Stroeve, P. (2012). Triggered release in lipid bilayer-capped mesoporous silica nanoparticles containing SPION using an alternating magnetic field. Chemical Communications, 48(45), 5647. doi:10.1039/c2cc31563gSancenón, F., Pascual, L., Oroval, M., Aznar, E., & Martínez-Máñez, R. (2015). Gated Silica Mesoporous Materials in Sensing Applications. ChemistryOpen, 4(4), 418-437. doi:10.1002/open.201500053Oroval, M., Climent, E., Coll, C., Eritja, R., Aviñó, A., Marcos, M. D., … Amorós, P. (2013). An aptamer-gated silica mesoporous material for thrombin detection. Chemical Communications, 49(48), 5480. doi:10.1039/c3cc42157kPascual, L., Baroja, I., Aznar, E., Sancenón, F., Marcos, M. D., Murguía, J. R., … Martínez-Máñez, R. (2015). Oligonucleotide-capped mesoporous silica nanoparticles as DNA-responsive dye delivery systems for genomic DNA detection. Chemical Communications, 51(8), 1414-1416. doi:10.1039/c4cc08306gGiménez, C., Climent, E., Aznar, E., Martínez-Máñez, R., Sancenón, F., Marcos, M. D., … Rurack, K. (2014). Über den chemischen Informationsaustausch zwischen gesteuerten Nanopartikeln. Angewandte Chemie, 126(46), 12838-12843. doi:10.1002/ange.201405580Llopis-Lorente, A., Díez, P., Sánchez, A., Marcos, M. D., Sancenón, F., Martínez-Ruiz, P., … Martínez-Máñez, R. (2017). Interactive models of communication at the nanoscale using nanoparticles that talk to one another. Nature Communications, 8(1). doi:10.1038/ncomms15511Lu, J., Liong, M., Li, Z., Zink, J. I., & Tamanoi, F. (2010). Biocompatibility, Biodistribution, and Drug-Delivery Efficiency of Mesoporous Silica Nanoparticles for Cancer Therapy in Animals. Small, 6(16), 1794-1805. doi:10.1002/smll.201000538Baeza, A., Manzano, M., Colilla, M., & Vallet-Regí, M. (2016). Recent advances in mesoporous silica nanoparticles for antitumor therapy: our contribution. Biomaterials Science, 4(5), 803-813. doi:10.1039/c6bm00039hRosenholm, J. M., Sahlgren, C., & Lindén, M. (2010). Towards multifunctional, targeted drug delivery systems using mesoporous silica nanoparticles – opportunities & challenges. Nanoscale, 2(10), 1870. doi:10.1039/c0nr00156bPoh, S., Lin, J. B., & Panitch, A. (2015). Release of Anti-inflammatory Peptides from Thermosensitive Nanoparticles with Degradable Cross-Links Suppresses Pro-inflammatory Cytokine Production. Biomacromolecules, 16(4), 1191-1200. doi:10.1021/bm501849pPatel, A., Cholkar, K., & Mitra, A. K. (2014). Recent developments in protein and peptide parenteral delivery approaches. Therapeutic Delivery, 5(3), 337-365. doi:10.4155/tde.14.5Sung, B., Kim, C., & Kim, M.-H. (2015). Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery. Journal of Colloid and Interface Science, 450, 26-33. doi:10.1016/j.jcis.2015.02.068Witting, M., Molina, M., Obst, K., Plank, R., Eckl, K. M., Hennies, H. C., … Hedtrich, S. (2015). Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules. Nanomedicine: Nanotechnology, Biology and Medicine, 11(5), 1179-1187. doi:10.1016/j.nano.2015.02.017Yu, E., Galiana, I., Martínez-Máñez, R., Stroeve, P., Marcos, M. D., Aznar, E., … Amorós, P. (2015). Poly(N-isopropylacrylamide)-gated Fe3O4/SiO2 core shell nanoparticles with expanded mesoporous structures for the temperature triggered release of lysozyme. Colloids and Surfaces B: Biointerfaces, 135, 652-660. doi:10.1016/j.colsurfb.2015.06.048Braun, K., Pochert, A., Lindén, M., Davoudi, M., Schmidtchen, A., Nordström, R., & Malmsten, M. (2016). 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The influence of P-glycoprotein expression and its inhibitors on the distribution of doxorubicin in breast tumors

Abstract Background Anti-cancer drugs access solid tumors via blood vessels, and must penetrate tumor tissue to reach all cancer cells. Previous studies have demonstrated steep gradients of decreasing doxorubicin fluorescence with increasing distance from blood vessels, such that many tumor cells are not exposed to drug. Studies using multilayered cell cultures show that increased P-glycoprotein (PgP) is associated with better penetration of doxorubicin, while PgP inhibitors decrease drug penetration in tumor tissue. Here we evaluate the effect of PgP expression on doxorubicin distribution in vivo. Methods Mice bearing tumor sublines with either high or low expression of PgP were treated with doxorubicin, with or without pre-treatment with the PgP inhibitors verapamil or PSC 833. The distribution of doxorubicin in relation to tumor blood vessels was quantified using immunofluorescence. Results Our results indicate greater uptake of doxorubicin by cells near blood vessels in wild type as compared to PgP-overexpressing tumors, and pre-treatment with verapamil or PSC 833 increased uptake in PgP-overexpressing tumors. However, there were steeper gradients of decreasing doxorubicin fluorescence in wild-type tumors compared to PgP overexpressing tumors, and treatment of PgP overexpressing tumors with PgP inhibitors led to steeper gradients and greater heterogeneity in the distribution of doxorubicin. Conclusion PgP inhibitors increase uptake of doxorubicin in cells close to blood vessels, have little effect on drug uptake into cells at intermediate distances, and might have a paradoxical effect to decrease doxorubicin uptake into distal cells. This effect probably contributes to the limited success of PgP inhibitors in clinical trials

Global-scale variations of the ratios of carbon to phosphorus in exported marine organic matter

The ratio of carbon (C) to phosphorus (P) in marine phytoplankton is thought to be constant throughout the worlds'oceans. Known as the Redfield ratio, this relationship describes the links between carbon and phosphorus cycling and marine ecosystems. However, variations in the stoichiometry of phytoplankton have recently been identified, in particular strong latitudinal variability. Here we assess the impact of this variability in the C:P ratio of biomass on the C:P ratio of organic matter that is exported to the deep ocean using a biogeochemical inverse-model based on a data-constrained ocean circulation model and a global database of dissolved inorganic carbon and phosphate measurements. We identify global patterns of variability in the C:P ratios of exported organic matter, with higher values in the nutrient-depleted subtropical gyres, where organic matter export is relatively low, and lower ratios in nutrient-rich upwelling zones and high-latitude regions, where organic matter export is high. This suggests that total carbon export is relatively constant throughout the oceans, in agreement with recent estimates of carbon fluxes. We conclude that the latitudinal patterns of C:P in exported organic matter are consistent with the large-scale stoichiometric variations in phytoplankton C:P. We suggest that a future expansion of nutrient-depleted waters could result in a shift to more efficient C export that compensates for the expected decline in productivity

Global rates of water-column denitrification derived from nitrogen gas measurements

Biologically available nitrogen (N) limits phytoplankton growth over much of the ocean. The rate at which N is removed from the contemporary ocean by denitrifying bacteria is highly uncertain 1-3. Some studies suggest that N losses exceed inputs 2,4-6; others argue for a balanced budget 3,7,8. Here, we use a global ocean circulation model to simulate the distribution of N 2 gas produced by denitrifying bacteria in the three main suboxic zones in the open ocean. By fitting the model to measured N 2 gas concentrations, we infer a globally integrated rate of water-column denitrification of 66 ±6 Tg N yr -1. Taking into account isotopic constraints on the fraction of denitrification occurring in the water column versus marine sediments, we estimate that the global rate of N loss from marine sediments and the oceanic water column combined amounts to around 230 ±60 Tg N yr -1. Given present estimates of N input rates, our findings imply a net loss of around 20 ± 70 Tg of N from the global ocean each year, indistinguishable from a balanced budget. A balanced N budget, in turn, implies that the marine N cycle is governed by strong regulatory feedbacks. © 2012 Macmillan Publishers Limited. All rights reserved

The population history of northeastern Siberia since the Pleistocene.

Northeastern Siberia has been inhabited by humans for more than 40,000 years but its deep population history remains poorly understood. Here we investigate the late Pleistocene population history of northeastern Siberia through analyses of 34 newly recovered ancient genomes that date to between 31,000 and 600 years ago. We document complex population dynamics during this period, including at least three major migration events: an initial peopling by a previously unknown Palaeolithic population of 'Ancient North Siberians' who are distantly related to early West Eurasian hunter-gatherers; the arrival of East Asian-related peoples, which gave rise to 'Ancient Palaeo-Siberians' who are closely related to contemporary communities from far-northeastern Siberia (such as the Koryaks), as well as Native Americans; and a Holocene migration of other East Asian-related peoples, who we name 'Neo-Siberians', and from whom many contemporary Siberians are descended. Each of these population expansions largely replaced the earlier inhabitants, and ultimately generated the mosaic genetic make-up of contemporary peoples who inhabit a vast area across northern Eurasia and the Americas

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

A Proof-Of-Principle Study of Epigenetic Therapy Added to Neoadjuvant Doxorubicin Cyclophosphamide for Locally Advanced Breast Cancer

BACKGROUND: Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine, plus the HDAC inhibitor magnesium valproate, were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy. METHODOLOGY: This was a single-arm interventional trial on breast cancer patients (ClinicalTrials.gov Identifier: NCT00395655). After signing informed consent, patients were typed for acetylator phenotype and then treated with hydralazine at 182 mg for rapid-, or 83 mg for slow-acetylators, and magnesium valproate at 30 mg/kg, starting from day –7 until chemotherapy ended, the latter consisting of four cycles of doxorubicin 60 mg/m(2) and cyclophosphamide 600 mg/m(2) every 21 days. Core-needle biopsies were taken from primary breast tumors at diagnosis and at day 8 of treatment with hydralazine and valproate. MAIN FINDINGS: 16 patients were included and received treatment as planned. All were evaluated for clinical response and toxicity and 15 for pathological response. Treatment was well-tolerated. The most common toxicity was drowsiness grades 1–2. Five (31%) patients had clinical CR and eight (50%) PR for an ORR of 81%. No patient progressed. One of 15 operated patients (6.6%) had pathological CR and 70% had residual disease <3 cm. There was a statistically significant decrease in global 5(m)C content and HDAC activity. Hydralazine and magnesium valproate up- and down-regulated at least 3-fold, 1,091 and 89 genes, respectively. CONCLUSIONS: Hydralazine and magnesium valproate produce DNA demethylation, HDAC inhibition, and gene reactivation in primary tumors. Doxorubicin and cyclophosphamide treatment is safe, well-tolerated, and appears to increase the efficacy of chemotherapy. A randomized phase III study is ongoing to support the efficacy of so-called epigenetic or transcriptional cancer therapy