Nasal Glucagon Reverses Insulin-induced Hypoglycemia With Less Rebound Hyperglycemia: Pooled Analysis of Clinical Trials.

Background: Rebound hyperglycemia may occur following glucagon treatment for severe hypoglycemia. We assessed rebound hyperglycemia occurrence after nasal glucagon (NG) or injectable glucagon (IG) administration in patients with type 1 diabetes (T1D) and type 2 diabetes (T2D). Methods: This was a pooled analysis of 3 multicenter, randomized, open-label studies (NCT03339453, NCT03421379, NCT01994746) in patients ≥18 years with T1D or T2D with induced hypoglycemia. Proportions of patients achieving treatment success [blood glucose (BG) increase to ≥70 mg/dL or increase of ≥20 mg/dL from nadir within 15 and 30 minutes]; BG ≥70 mg/dL within 15 minutes; in-range BG (70-180 mg/dL) 1 to 2 and 1 to 4 hours postdose; and BG >180 mg/dL 1 to 2 and 1 to 4 hours postdose were compared. Incremental area under curve (iAUC) of BG >180 mg/dL and area under curve (AUC) of observed BG values postdose were analyzed. Safety was assessed in all studies. Results: Higher proportions of patients had in-range BG with NG vs IG (1-2 hours: P = .0047; 1-4 hours: P = .0034). Lower proportions of patients had at least 1 BG value >180 mg/dL with NG vs IG (1-2 hours: P = .0034; 1-4 hours: P = .0068). iAUC and AUC were lower with NG vs IG (P = .025 and P < .0001). As expected, similar proportions of patients receiving NG or IG achieved treatment success at 15 and 30 minutes (97-100%). Most patients had BG ≥70 mg/dL within 15 minutes (93-96%). The safety profile was consistent with previous studies. Conclusion: This study demonstrated lower rebound hyperglycemia risk after NG treatment compared with IG. Clinical Trial Registration: NCT03421379, NCT03339453, NCT01994746.

Unique evolution of foraminiferal calcification to survive global changes.

Foraminifera, the most ancient known calcium carbonate-producing eukaryotes, are crucial players in global biogeochemical cycles and well-used environmental indicators in biogeosciences. However, little is known about their calcification mechanisms. This impedes understanding the organismal responses to ocean acidification, which alters marine calcium carbonate production, potentially leading to biogeochemical cycle changes. We conducted comparative single-cell transcriptomics and fluorescent microscopy and identified calcium ion (Ca2+) transport/secretion genes and α-carbonic anhydrases that control calcification in a foraminifer. They actively take up Ca2+ to boost mitochondrial adenosine triphosphate synthesis during calcification but need to pump excess intracellular Ca2+ to the calcification site to prevent cell death. Unique α-carbonic anhydrase genes induce the generation of bicarbonate and proton from multiple CO2 sources. These control mechanisms have evolved independently since the Precambrian to enable the development of large cells and calcification despite decreasing Ca2+ concentrations and pH in seawater. The present findings provide previously unknown insights into the calcification mechanisms and their subsequent function in enduring ocean acidification.

Dose Rationale of Nasal Glucagon in Japanese Pediatric Patients with Diabetes Using Pharmacokinetic/Pharmacodynamic Modeling and Simulation.

BACKGROUND: Nasal glucagon (NG) 3 mg is approved in Japan to treat hypoglycemia in pediatric patients with diabetes, but an NG clinical study has not been performed in Japanese children because of practical and ethical concerns. OBJECTIVE: The aim of this study is to support the dose rationale for NG 3 mg in Japanese pediatric patients with diabetes using modeling and simulation. METHODS: We used a pharmacokinetic/pharmacodynamic bridging approach to extrapolate the available clinical data to Japanese pediatric patients. Population pharmacokinetic/pharmacodynamic modeling was performed using data from seven clinical studies, including five studies in non-Japanese adults, one study in Japanese adults, and one study in non-Japanese pediatric patients. Simulation was then used to estimate glucagon exposure and glucose response after NG 3-mg administration for three age categories of Japanese pediatric patients: 4 to < 8, 8 to < 12, and 12 to < 18 years. Treatment success was defined as an increase in blood glucose to ≥ 70 or ≥ 20 mg/dL from nadir within 30 min after administration of NG 3 mg. Safety was assessed in relation to the predicted maximum glucagon concentration of NG 3 mg using NG clinical trial data and published data on intravenous and intramuscular glucagon. RESULTS: The data showed a rapid and robust glucose response following NG 3 mg in Japanese and non-Japanese adults and non-Japanese pediatric patients, with some differences in glucagon exposure observed across studies. The pharmacokinetic/pharmacodynamic model described the observed clinical data well, and simulations indicated that > 99% of hypoglycemic Japanese pediatric patients in all three age groups would achieve treatment success. Predicted glucose responses to NG 3 mg in Japanese pediatric patients were comparable to those of intramuscular glucagon. Maximum concentration was not associated with the occurrence and severity of common adverse events (nausea, vomiting, and headache) in NG clinical studies. Furthermore, the predicted maximum concentration in Japanese pediatric patients, despite being higher than the observed maximum concentration in NG clinical studies, was substantially lower than the observed maximum concentration of 1 mg of intravenous glucagon, without serious safety issues. CONCLUSIONS: This analysis suggests NG 3 mg has robust efficacy without serious safety concerns in Japanese pediatric patients with diabetes.

Multimodal image and spectral feature learning for efficient analysis of water-suspended particles.

We have developed a method to combine morphological and chemical information for the accurate identification of different particle types using optical measurement techniques that require no sample preparation. A combined holographic imaging and Raman spectroscopy setup is used to gather data from six different types of marine particles suspended in a large volume of seawater. Unsupervised feature learning is performed on the images and the spectral data using convolutional and single-layer autoencoders. The learned features are combined, where we demonstrate that non-linear dimensional reduction of the combined multimodal features can achieve a high clustering macro F1 score of 0.88, compared to a maximum of 0.61 when only image or spectral features are used. The method can be applied to long-term monitoring of particles in the ocean without the need for sample collection. In addition, it can be applied to data from different types of sensor measurements without significant modifications.

Polymer-coated carbon nanotube hybrids with functional peptides for gene delivery into plant mitochondria.

The delivery of genetic material into plants has been historically challenging due to the cell wall barrier, which blocks the passage of many biomolecules. Carbon nanotube-based delivery has emerged as a promising solution to this problem and has been shown to effectively deliver DNA and RNA into intact plants. Mitochondria are important targets due to their influence on agronomic traits, but delivery into this organelle has been limited to low efficiencies, restricting their potential in genetic engineering. This work describes the use of a carbon nanotube-polymer hybrid modified with functional peptides to deliver DNA into intact plant mitochondria with almost 30 times higher efficiency than existing methods. Genetic integration of a folate pathway gene in the mitochondria displays enhanced plant growth rates, suggesting its applications in metabolic engineering and the establishment of stable transformation in mitochondrial genomes. Furthermore, the flexibility of the polymer layer will also allow for the conjugation of other peptides and cargo targeting other organelles for broad applications in plant bioengineering.

The coral reef-dwelling Peneroplis spp. shows calcification recovery to ocean acidification conditions.

Large Benthic Foraminifera are a crucial component of coral-reef ecosystems, which are currently threatened by ocean acidification. We conducted culture experiments to evaluate the impact of low pH on survival and test dissolution of the symbiont-bearing species Peneroplis spp., and to observe potential calcification recovery when specimens are placed back under reference pH value (7.9). We found that Peneroplis spp. displayed living activity up to 3 days at pH 6.9 (Ωcal < 1) or up to 1 month at pH 7.4 (Ωcal > 1), despite the dark and unfed conditions. Dissolution features were observed under low Ωcal values, such as changes in test density, peeled extrados layers, and decalcified tests with exposed organic linings. A new calcification phase started when specimens were placed back at reference pH. This calcification's resumption was an addition of new chambers without reparation of the dissolved parts, which is consistent with the porcelaneous calcification pathway of Peneroplis spp. The most decalcified specimens displayed a strong survival response by adding up to 8 new chambers, and the contribution of food supply in this process was highlighted. These results suggest that porcelaneous LBF species have some recovery abilities to short exposure (e.g., 3 days to 1 month) to acidified conditions. However, the geochemical signature of trace elements in the new calcite was impacted, and the majority of the new chambers were distorted and resulted in abnormal tests, which might hinder the specimens' reproduction and thus their survival on the long term.

Micro- and nanometric characterization of the celestite skeleton of acantharian species (Radiolaria, Rhizaria).

We clarified the specific micrometric arrangement and nanometric structure of the radiolarian crystalline spines that are not a simple single crystal. A body of the celestite (SrSO4) skeleton of acantharian Acanthometra cf. multispina (Acanthometridae) composed of 20 radial spines having four blades was characterized using microfocus X-ray computed tomography. The regular arrangement of three types of spines was clarified with the connection of the blades around the root of each spine. The surface of the spines was covered with a chitin-based organic membrane to prevent from dissolution in seawater. In the nanometric scale, the mesocrystalline structure that consists of nanoscale grains having distorted single-crystal nature was revealed using scanning- and transmission electron microscopies, electron diffraction, and Raman spectroscopy. The acantharian skeletons have a crystallographically controlled architecture that is covered with a protective organic membrane. These facts are important for penetrating the nature of biogenic minerals.

Conversations and Reactions Around Severe Hypoglycemia (CRASH): Japan Results From a Global Survey of People with T1DM or Insulin-Treated T2DM and Caregivers.

AIMS: The CRASH study examined severe hypoglycemia (SH) experiences among people with diabetes (PWD) and caregivers across eight countries. Here we report findings from the Japan cohort, with references to data from the United Kingdom (UK) cohort. MATERIALS AND METHODS: Adults with type 1 (T1DM) or insulin-treated type 2 diabetes mellitus (T2DM) and caregivers (not necessarily related) were recruited from online patient panels. Participants who had experienced at least one SH event in the past 3 years were eligible for study inclusion. Participants completed an online survey regarding their experience with SH, its treatment, and actions during and after an event. RESULTS: Of the 9367 PWD and caregivers from the online patient panels, 8475 participants were ineligible and a total of 53 Japanese participants (35 T1DM, 9 T2DM, 9 caregivers) completed the survey. Most SH incidents occurred at home and were unattended by a healthcare provider. For T1DM, 29% of Japan PWD and 13% of the UK PWD called an ambulance during an SH event; of these, 90% (Japan) and 50% (UK) were transported to hospital. Glucagon use was low (3% Japan and 10% UK for T1DM). Japanese respondents reported emotional impacts of SH, including feeling scared (86% T1DM, 56% T2DM), unprepared (63% T1DM, 78% T2DM), and helpless (60% T1DM, 33% T2DM). Despite the emotional burden, most PWD did not immediately discuss their SH event with a healthcare provider, with the majority (75% T1DM, 71% T2DM) waiting until their next doctor's appointment. CONCLUSION: Conversations around SH between healthcare providers and PWD appear to be insufficient in Japan. An emotional burden of SH was reported by PWD and caregivers. Education regarding the prevention of SH and available treatment options may reduce SH events and improve treatment preparation, while alleviating PWD concerns.

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Antibody-Conjugated Gel-Coated Single-Walled Carbon Nanotubes as Photothermal Agents.

Photothermal therapy (PTT) using near-infrared (NIR) light is an attractive treatment modality for cancer, in which photothermal agents absorb energy from photons and convert it into thermal energy to lead to cancer cell death. Among the various organic and inorganic materials, single-walled carbon nanotubes (SWCNTs) are promising candidates for NIR photothermal agents due to their strong absorption in this region as well as their high photothermal conversion efficiency. In the development of the SWCNT-based PTT materials, modifications of SWCNTs to offer a stable dispersion for biocompatibility as well as to target the tumor of choice while maintaining their NIR absorption have been required. While modification of SWCNTs through noncovalent methods can be achieved, these modifications can be easily reversed in the body. Contrarily, modifications through covalent attachments, while more desirable, may compromise the NIR absorption characteristics of the SWCNTs. Previously, we reported the development of a synthetic strategy to coat SWCNTs with a cross-linked polymer (i.e., a gel) through a process called CNT Micelle Polymerization and successfully introduced maleimide groups that allowed for postmodification through the ene-thiol reaction without deteriorating the NIR absorption. In this report, we postmodify thiol-containing antibodies (anti-TRP-1, a melanoma specific protein) using maleimide chemistry and find that the SWCNTs conjugated with anti-TRP-1 maintain the characteristic NIR absorption as SWCNTs with dispersion stability. It is estimated that 50 maleimide groups are incorporated in one SWCNT (ca. 280 nm long) and they are modified with 32 TRP-1 fragments. Finally, we successfully use these targeted SWCNTs for the PTT of the melanoma cell line using NIR light (1064 nm; 2 W, 5 min). Our method can be extended to a vast array of specific antibodies as well as other targeting agents.

Nasal glucagon as a viable alternative for treating insulin-induced hypoglycaemia in Japanese patients with type 1 or type 2 diabetes: A phase 3 randomized crossover study.

AIM: To compare nasal glucagon (NG) with intramuscular glucagon (IMG) for the treatment of insulin-induced hypoglycaemia in Japanese patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: This phase 3, randomized, open-label, two-treatment, two-period crossover non-inferiority study enrolled Japanese adults with T1DM or T2DM on insulin therapy, with glycated haemoglobin levels ≤86 mmol/mol (≤10%). After ≥8 hours of fasting, hypoglycaemia was induced with human regular insulin (intravenous infusion). Patients received NG 3 mg or IMG 1 mg approximately 5 minutes after insulin termination. The primary endpoint was the proportion of patients achieving treatment success [plasma glucose (PG) increase to ≥3.9 mmol/L (≥70 mg/dL) or ≥1.1 mmol/L (≥20 mg/dL) increase from the PG nadir within 30 minutes of receiving glucagon]. Non-inferiority was declared if the upper limit of the two-sided 95% confidence interval (CI) of the mean difference in the percentage of patients achieving treatment success (IMG minus NG) was <10%. RESULTS: Seventy-five patients with T1DM (n = 34) or T2DM (n = 41) were enrolled; 72 patients (50 men, 22 women) received ≥1 study drug dose (T1DM, n = 33; T2DM, n = 39). Sixty-eight patients completed the study and were evaluable. All NG- and IMG-treated patients achieved treatment success (treatment arm difference: 0%; upper limit of two-sided 95% CI 1.47%); NG met prespecified conditions defining non-inferiority versus IMG. Glucagon was rapidly absorbed after both nasal and intramuscular administration; PG profiles were similar between administration routes during the first 60 minutes post dose. Study drug-related treatment-emergent adverse events affecting >2 patients were rhinalgia, increased blood pressure, nausea, ear pain and vomiting in the NG group, and nausea and vomiting in the IMG group. CONCLUSION: Nasal glucagon was non-inferior to IMG for successful treatment of insulin-induced hypoglycaemia in Japanese patients with T1DM/T2DM, supporting use of NG as a rescue treatment for severe hypoglycaemia.

Guanine crystals regulated by chitin-based honeycomb frameworks for tunable structural colors of sapphirinid copepod, Sapphirina nigromaculata.

Sapphirinid copepods, which are marine zooplankton, exhibit tunable structural colors originating from a layered structure of guanine crystal plates. In the present study, the coloring portion of adult male of a sapphirinid copepod, Sapphirina nigromaculata, under the dorsal body surface was characterized to clarify the regulation and actuation mechanism of the layered guanine crystals for spectral control. The coloring portions are separated into small domains 70-100 µm wide consisting of an ordered array of stacked hexagonal plates ~1.5 µm wide and ~80 nm thick. We found the presence of chitin-based honeycomb frameworks that are composed of flat compartments regulating the guanine crystal plates. The structural color is deduced to be tuned from blue to achromatic via yellow and purple by changing the interplate distance according to vital observation and optical simulation using a photonic array model. The framework structures are essential for the organization and actuation of the particular photonic arrays for the exhibition of the tunable structural color.

Usability of Nasal Glucagon Device: Partially Randomized Caregiver and Third-Party User Experience Trial with Simulated Administration at a Japanese Site.

INTRODUCTION: Glucagon is the only approved medicine for severe hypoglycemia available for caregivers of people with diabetes. Nasal glucagon (NG) was recently approved in the USA as a needle-free, ready-to-use alternative to injectable glucagon. This simulated user experience study in Japan compared NG and intramuscular glucagon (IMG) administration by caregivers, and NG administration by untrained third parties. METHODS: This was an open-label, single-center, partially randomized crossover, simulated user experience trial conducted in Japan (October 2018 to December 2018). Caregivers who live with and care for a relative with diabetes were randomized (1:1, stratified by patient diabetes type 1 or 2) to one of two simulated administration sequences (group 1: NG then IMG; group 2: IMG then NG). Caregivers received training on each device 2 weeks before simulated administration of the device. Third parties received no training and only conducted simulated NG administration. Outcome measures included the percentage of successful administrations (based on critical step completion and dose; primary outcome), time to complete administration, and user satisfaction/preferences. RESULTS: In caregivers (N = 19), the percentage of successful administrations was greater (89.5% vs 26.3%, P < 0.001) and mean time to complete administration was shorter (23.9 vs 207.3 s, P < 0.001) with NG than with IMG. In third parties (N = 20), 95% of NG administration attempts were successful (mean time to complete administration, 55.5 s). All caregivers and 80% of third parties reported that the NG device was easy to use. All caregivers and 70% of third parties were confident and willing to use the device in a real emergency, and more than 80% of caregivers preferred the NG device to IMG. CONCLUSION: This simulated user experience study confirmed that glucagon administration using a nasal delivery device was quicker, easier, and had a higher success rate than intramuscular administration in Japan, where the glucagon injection kit is not available. FUNDING: Eli Lilly. Plain language summary available for this article.

Complex factors shape phenotypic variation in deep-sea limpets.

Pectinodontid limpets are important members of deep-sea hot vents and cold seeps as can be seen by their conspicuous presence in both extant and extinct systems. They have traditionally been classified into different genera and species based on shell and radula characteristics; the reliability of these characters has been questioned but not tested thoroughly. Here, for the first time in taxa endemic to deep-sea chemosynthetic ecosystems, we combine substrate translocation with molecular data to assess the plasticity and variability of key phenotypic characters. Molecular data revealed that several 'species' of extant vent/seep pectinodontids actually represent intergrading morphotypes of a single, highly plastic, evolutionary lineage, with each morphological trait being possibly influenced differently by environmental and genetic factors. Our results challenge previous interpretations of paleoecology at fossil chemosynthetic ecosystems and highlight the importance of modern analogues in understanding fossil systems.

Identification of cells expressing two peptidoglycan recognition proteins in the gill of the vent mussel, Bathymodiolus septemdierum.

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

Brighter near-IR emission of single-walled carbon nanotubes modified with a cross-linked polymer coating.

Photoluminescence (PL) in the near-infrared (NIR) region is an attractive feature of single-walled carbon nanotubes (SWNTs). In this study, we investigated the effect of the chemical structure of the cross-linked polymer coating of polymer-coated SWNTs on the NIR PL emission intensity. We found that brighter NIR emission can be achieved using a more hydrophobic polymer coating.

Compositional and Functional Shifts in the Epibiotic Bacterial Community of Shinkaia crosnieri Baba & Williams (a Squat Lobster from Hydrothermal Vents) during Methane-Fed Rearing.

The hydrothermal vent squat lobster Shinkaia crosnieri Baba & Williams harbors an epibiotic bacterial community, which is numerically and functionally dominated by methanotrophs affiliated with Methylococcaceae and thioautotrophs affiliated with Sulfurovum and Thiotrichaceae. In the present study, shifts in the phylogenetic composition and metabolic function of the epibiont community were investigated using S. crosnieri individuals, which were reared for one year in a tank fed with methane as the energy and carbon source. The results obtained indicated that indigenous predominant thioautotrophic populations, such as Sulfurovum and Thiotrichaceae members, became absent, possibly due to the lack of an energy source, and epibiotic communities were dominated by indigenous Methylococcaceae and betaproteobacterial methylotrophic members that adapted to the conditions present during rearing for 12 months with a supply of methane. Furthermore, the overall phylogenetic composition of the epibiotic community markedly changed from a composition dominated by chemolithotrophs to one enriched with cross-feeding heterotrophs in addition to methanotrophs and methylotrophs. Thus, the composition and function of the S. crosnieri epibiotic bacterial community were strongly affected by the balance between the energy and carbon sources supplied for chemosynthetic production as well as that between the production and consumption of organic compounds.

Synthesis of Single-Walled Carbon Nanotubes Coated with Thiol-Reactive Gel via Emulsion Polymerization.

Single-walled carbon nanotubes (SWNTs) have unique near-infrared absorption and photoemission properties that are attractive for in vivo biological applications such as photothermal cancer treatment and bioimaging. Therefore, a smart functionalization strategy for SWNTs to create biocompatible surfaces and introduce various ligands to target active cancer cells without losing the unique optical properties of the SWNTs is strongly desired. This paper reports the design and synthesis of a SWNT/gel hybrid containing maleimide groups, which react with various thiol compounds through Michael addition reactions. In this hybrid, the method called carbon nanotube micelle polymerization was used to noncovalently modify the surface of SWNTs with a cross-linked polymer gel layer. This method can form an extremely stable gel layer on SWNTs; such stability is essential for in vivo biological applications. The monomer used to form the gel layer contained a maleimide group, which was protected with furan in endo-form. The resulting hybrid was treated in water to induce deprotection via a retro-Diels-Alder reaction and then functionalized with thiol compounds through Michael addition. The functionalization of the hybrid was explored using a thiol-containing fluorescent dye as a model thiol, and the formation of the SWNT-dye conjugate was confirmed by energy transfer from the dye to SWNTs. Our strategy offers a promising SWNT-based platform for biological functionalization for cancer targeting, imaging, and treatment.

Decalcification and survival of benthic foraminifera under the combined impacts of varying pH and salinity.

Coastal areas display natural large environmental variability such as frequent changes in salinity, pH, and carbonate chemistry. Anthropogenic impacts - especially ocean acidification - increase this variability, which may affect the living conditions of coastal species, particularly, calcifiers. We performed culture experiments on living benthic foraminifera to study the combined effects of lowered pH and salinity on the calcification abilities and survival of the coastal, calcitic species Ammonia sp. and Elphidium crispum. We found that in open ocean conditions (salinity ∼35) and lower pH than usual values for these species, the specimens displayed resistance to shell (test) dissolution for a longer time than in brackish conditions (salinity ∼5 to 20). However, the response was species specific as Ammonia sp. specimens survived longer than E. crispum specimens when placed in the same conditions of salinity and pH. Living, decalcified juveniles of Ammonia sp. were observed and we show that desalination is one cause for the decalcification. Finally, we highlight the ability of foraminifera to survive under Ωcalc < 1, and that high salinity and [Ca2+] as building blocks are crucial for the foraminiferal calcification process.

Proton pumping accompanies calcification in foraminifera.

Ongoing ocean acidification is widely reported to reduce the ability of calcifying marine organisms to produce their shells and skeletons. Whereas increased dissolution due to acidification is a largely inorganic process, strong organismal control over biomineralization influences calcification and hence complicates predicting the response of marine calcifyers. Here we show that calcification is driven by rapid transformation of bicarbonate into carbonate inside the cytoplasm, achieved by active outward proton pumping. Moreover, this proton flux is maintained over a wide range of pCO2 levels. We furthermore show that a V-type H+ ATPase is responsible for the proton flux and thereby calcification. External transformation of bicarbonate into CO2 due to the proton pumping implies that biomineralization does not rely on availability of carbonate ions, but total dissolved CO2 may not reduce calcification, thereby potentially maintaining the current global marine carbonate production.