Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

The Role of Cell Sterilization in Population Based Studies of Radiogenic Second Cancers Following Radiation Therapy

Advances in radiotherapy have generated increased interest in comparative studies of treatment techniques and their effectiveness. In this respect, pediatric patients are of specific interest because of their sensitivity to radiation induced second cancers. However, due to the rarity of childhood cancers and the long latency of second cancers, large sample sizes are unavailable for the epidemiological study of contemporary radiotherapy treatments. Additionally, when specific treatments are considered, such as proton therapy, sample sizes are further reduced due to the rareness of such treatments. We propose a method to improve statistical power in micro clinical trials. Specifically, we use a more biologically relevant quantity, cancer equivalent dose (DCE), to estimate risk instead of mean absorbed dose (DMA). Our objective was to demonstrate that when DCE is used fewer subjects are needed for clinical trials. Thus, we compared the impact of DCE vs. DMA on sample size in a virtual clinical trial that estimated risk for second cancer (SC) in the thyroid following craniospinal irradiation (CSI) of pediatric patients using protons vs. photons. Dose reconstruction, risk models, and statistical analysis were used to evaluate SC risk from therapeutic and stray radiation from CSI for 18 patients. Absorbed dose was calculated in two ways: with (1) traditional DMA and (2) with DCE. DCE and DMA values were used to estimate relative risk of SC incidence (RRCE and RRMA, respectively) after proton vs. photon CSI. Ratios of RR for proton vs. photon CSI (RRRCE and RRRMA) were then used in comparative estimations of sample size to determine the minimal number of patients needed to maintain 80% statistical power when using DCE vs. DMA. For all patients, we found that protons substantially reduced the risk of developing a second thyroid cancer when compared to photon therapy. Mean RRR values were 0.052±0.014 and 0.087±0.021 for RRRMA and RRRCE, respectively. However, we did not find that use of DCE reduced the number of patents needed for acceptable statistical power (i.e, 80%). In fact, when considerations were made for RRR values that met equipoise requirements and the need for descriptive statistics, the minimum number of patients needed for a micro-clinical trial increased from 17 using DMA to 37 using DCE. Subsequent analyses revealed that for our sample, the most influential factor in determining variations in sample size was the experimental standard deviation of estimates for RRR across the patient sample. Additionally, because the relative uncertainty in dose from proton CSI was so much larger (on the order of 2000 times larger) than the other uncertainty terms, it dominated the uncertainty in RRR. Thus, we found that use of corrections for cell sterilization, in the form of DCE, may be an important and underappreciated consideration in the design of clinical trials and radio-epidemiological studies. In addition, the accurate application of cell sterilization to thyroid dose was sensitive to variations in absorbed dose, especially for proton CSI, which may stem from errors in patient positioning, range calculation, and other aspects of treatment planning and delivery.

Development of dendritic tonic GABAergic inhibition regulates excitability and plasticity in CA1 pyramidal neurons

Synaptic plasticity rules change during development: while hippocampal synapses can be potentiated by a single action potential pairing protocol in young neurons, mature neurons require burst firing to induce synaptic potentiation. An essential component for spike timing-dependent plasticity is the backpropagating action potential (BAP). BAP along the dendrites can be modulated by morphology and ion channel composition, both of which change during late postnatal development. However it is unclear whether these dendritic changes can explain the developmental changes in synaptic plasticity induction rules. Here, we show that tonic GABAergic inhibition regulates dendritic action potential backpropagation in adolescent but not pre-adolescent CA1 pyramidal neurons. These developmental changes in tonic inhibition also altered the induction threshold for spike timing-dependent plasticity in adolescent neurons. This GABAergic regulatory effect upon backpropagation is restricted to distal regions of apical dendrites (>200 μm) and mediated by α5-containing GABA(A) receptors. Direct dendritic recordings demonstrate α5-mediated tonic GABA(A) currents in adolescent neurons which can modulate backpropagating action potentials. These developmental modulations in dendritic excitability could not be explained by concurrent changes in dendritic morphology. To explain our data, model simulations propose a distally-increasing or localized distal expression of dendritic α5 tonic inhibition in mature neurons. Overall, our results demonstrate that dendritic integration and plasticity in more mature dendrites are significantly altered by tonic α5 inhibition in a dendritic region-specific and developmentally-regulated manner.

Seasonal Variation in the Capacity for Plant Trait Measures to Predict Grassland Carbon and Water Fluxes

There is a need for accurate predictions of ecosystem carbon (C) and water fluxes in field conditions. Previous research has shown that ecosystem properties can be predicted from community abundance-weighted means (CWM) of plant functional traits and measures of trait variability within a community (FDvar). The capacity for traits to predict carbon (C) and water fluxes, and the seasonal dependency of these trait-function relationships has not been fully explored. Here we measured daytime C and water fluxes over four seasons in grasslands of a range of successional ages in southern England. In a model selection procedure, we related these fluxes to environmental covariates and plant biomass measures before adding CWM and FDvar plant trait measures that were scaled up from measures of individual plants grown in greenhouse conditions. Models describing fluxes in periods of low biological activity contained few predictors, which were usually abiotic factors. In more biologically active periods, models contained more predictors, including plant trait measures. Field-based plant biomass measures were generally better predictors of fluxes than CWM and FDvar traits. However, when these measures were used in combination traits accounted for additional variation. Where traits were significant predictors their identity often reflected seasonal vegetation dynamics. These results suggest that database derived trait measures can improve the prediction of ecosystem C and water fluxes. Controlled studies and those involving more detailed flux measurements are required to validate and explore these findings, a worthwhile effort given the potential for using simple vegetation measures to help predict landscape-scale fluxes.

Use of contrast-enhanced fluid-attenuated inversion recovery sequence to detect brain lesions in dogs and cats.

BACKGROUND The diagnostic value of a contrast-enhanced T2-weighted FLAIR sequence (ceFLAIR) in brain imaging is unclear. HYPOTHESIS/OBJECTIVES That the number of brain lesions detected with ceFLAIR would be no greater than the sum of lesions detected with nFLAIR and ceT1W sequence. ANIMALS One hundred and twenty-nine animals (108 dogs and 21 cats) undergoing magnetic resonance imaging (MRI) of the head between July 2010 and October 2011 were included in the study. METHODS A transverse ceFLAIR was added to a standard brain MRI protocol. Presence and number of lesions were determined based on all available MRI sequences by 3 examiners in consensus and lesion visibility was evaluated for nFLAIR, ceFLAIR, and ceT1W sequences. RESULTS Eighty-three lesions (58 intra-axial and 25 extra-axial) were identified in 51 patients. Five lesions were detected with nFLAIR alone, 2 with ceT1W alone, and 1 with ceFLAIR alone. Significantly higher numbers of lesions were detected using ceFLAIR than nFLAIR (76 versus 67 lesions; P = 0.04), in particular for lesions also detected with ceT1W images (53 versus 40; P =.01). There was no significant difference between the number of lesions detected with combined nFLAIR and ceT1W sequences compared to those detected with ceFLAIR (82 versus 76; P =.25). CONCLUSION AND CLINICAL IMPORTANCE Use of ceFLAIR as a complementary sequence to nFLAIR and ceT1W sequences did not improve the detection of brain lesions and cannot be recommended as part of a routine brain MRI protocol in dogs and cats with suspected brain lesions.

Cardiac lipid levels show diurnal changes and long-term variations in healthy human subjects

(1) H-MRS is regularly applied to determine lipid content in ectopic tissue - mostly skeletal muscle and liver - to investigate physiological and/or pathologic conditions, e.g. insulin resistance. Technical developments also allow non-invasive in vivo assessment of cardiac lipids; however, basic data about methodological reliability (repeatability) and physiological variations are scarce. The aim of the presented work was to determine potential diurnal changes of cardiac lipid stores in humans, and to put the results in relation to methodological repeatability and normal physiological day-to-day variations. Optimized cardiac- and respiratory-gated (1) H-MRS was used for non-invasive quantification of intracardiomyocellular lipids (ICCL), creatine, trimethyl-ammonium compounds (TMA), and taurine in nine healthy young men at three time points per day on two days separated by one week. This design allowed determination of (a) diurnal changes, (b) physiological variation over one week and (c) methodological repeatability of the ICCL levels. Comparison of fasted morning to post-absorptive evening measurements revealed a significant 37 ± 19% decrease of ICCL during the day (p = 0.0001). There was a significant linear correlation between ICCL levels in the morning and their decrease during the day (p = 0.015). Methodological repeatability for the ICCL/creatine ratio was excellent, with a coefficient of variance of ~5%, whereas physiological variation was found to be considerably higher (22%) in spite of a standardized physiological preparation protocol. In contrast, TMA levels remained stable over this time period. The proposed (1) H-MRS technique provides a robust way to investigate relevant physiological changes in cardiac metabolites, in particular ICCL. The present results suggest that ICCL reveal a diurnal course, with higher levels in the morning as compared to evening. In addition, a considerable long-term variation of ICCL levels, in both the morning and evening, was documented. Given the high methodological repeatability, these effects should be taken into account in studies investigating the metabolic role of ICCL.

Zinc supplementation reduced DNA breaks in Ethiopian women.

Assessment of zinc status remains a challenge largely because serum/plasma zinc may not accurately reflect an individual's zinc status. The comet assay, a sensitive method capable of detecting intracellular DNA strand breaks, may serve as a functional biomarker of zinc status. We hypothesized that effects of zinc supplementation on intracellular DNA damage could be assessed from samples collected in field studies in Ethiopia using the comet assay. Forty women, from villages where reported consumption of meat was less than once per month and phytate levels were high, received 20 mg zinc as zinc sulfate or placebo daily for 17 days in a randomized placebo-controlled trial. Plasma zinc concentrations were determined by inductively coupled plasma mass spectrometry. Cells from whole blood at the baseline and end point of the study were embedded in agarose, electrophoresed, and stained before being scored by an investigator blinded to the treatments. Although zinc supplementation did not significantly affect plasma zinc, mean (± SEM) comet tail moment measurement of supplemented women decreased from 39.7 ± 2.7 to 30.0 ± 1.8 (P< .005), indicating a decrease in DNA strand breaks in zinc-supplemented individuals. These findings demonstrated that the comet assay could be used as a functional assay to assess the effects of zinc supplementation on DNA integrity in samples collected in a field setting where food sources of bioavailable zinc are limited. Furthermore, the comet assay was sufficiently sensitive to detect changes in zinc status as a result of supplementation despite no significant changes in plasma zinc.

Towards Using Microstate-Neurofeedback for the Treatment of Psychotic Symptoms in Schizophrenia. A Feasibility Study in Healthy Participants

Spontaneous EEG signal can be parsed into sub-second periods of stable functional states (microstates) that assumingly correspond to brief large scale synchronization events. In schizophrenia, a specific class of microstate (class "D") has been found to be shorter than in healthy controls and to be correlated with positive symptoms. To explore potential new treatment options in schizophrenia, we tested in healthy controls if neurofeedback training to self-regulate microstate D presence is feasible and what learning patterns are observed. Twenty subjects underwent EEG-neurofeedback training to up-regulate microstate D presence. The protocol included 20 training sessions, consisting of baseline trials (resting state), regulation trials with auditory feedback contingent on microstate D presence, and a transfer trial. Response to neurofeedback was assessed with mixed effects modelling. All participants increased the percentage of time spent producing microstate D in at least one of the three conditions (p < 0.05). Significant between-subjects across-sessions results showed an increase of 0.42 % of time spent producing microstate D in baseline (reflecting a sustained change in the resting state), 1.93 % of increase during regulation and 1.83 % during transfer. Within-session analysis (performed in baseline and regulation trials only) showed a significant 1.65 % increase in baseline and 0.53 % increase in regulation. These values are in a range that is expected to have an impact upon psychotic experiences. Additionally, we found a negative correlation between alpha power and microstate D contribution during neurofeedback training. Given that microstate D has been related to attentional processes, this result provides further evidence that the training was to some degree specific for the attentional network. We conclude that microstate-neurofeedback training proved feasible in healthy subjects. The implementation of the same protocol in schizophrenia patients may promote skills useful to reduce positive symptoms by means of EEG-neurofeedback.

Management of a complication with a fractured zirconia implant abutment in the esthetic zone

Technical complications in implant prosthetic cases represent a major challenge in dentistry. This case report describes minimally invasive management to recover an implant with a fractured remnant of a zirconia abutment, including provisional rehabilitation during a sequential treatment protocol in the esthetic zone. A patient was treated with a screw-retained one-piece implant-supported reconstruction made of a customized zirconia abutment with direct ceramic veneering in the maxillary right central incisor position. During the prosthetic try-in, a fracture in the apical portion of the abutment was evident. The first rescue attempt led to fracture of the retrieval instrument. Immediately, an individualized wired construction was applied to bond the existing fractured reconstruction to the neighboring teeth to maintain the peri-implant mucosal architecture. Because the implant screw canal was blocked, a customized round bur had to be manufactured and was placed in the implant axis with a specific bracket tool from the service set to protect the interior implant threads. Then, the drills of the service set were guided by the newly created access to remove the fractured remnants. The implant screw was retapped and the area rinsed with chlorhexidine solution. All remnants were removed without the need for surgical intervention. Neither the implant connection nor the bone-to-implant interface was damaged. The stepwise treatment approach with the customized round bur combined with the system-specific drills of the service set saved the blocked implant so that the patient could be successfully rehabilitated with a new implant reconstruction.

Linking soil properties and pre-Columbian agricultural strategies in the Bolivian lowlands: The case of raised fields in Exaltación

This paper aims to further our understanding of pre-Columbian agricultural systems in the Llanos de Moxos, Bolivia. Three different types of raised fields co-existing in the same site near the community of Exaltación, in north-western Beni, were studied. The morphology, texture and geochemistry of the soils of these fields and the surrounding area were analysed. Differences in field design have often been associated with the diversity of cultural practices. Our results suggest that in the study area differences in field shape, height and layout are primarily the result of an adaptation to the local edaphology. By using the technology of raised fields, pre-Columbian people were able to drain and cultivate soils with very different characteristics, making the land suitable for agriculture and possibly different crops. This study also shows that some fields in the Llanos de Moxos were built to prolong the presence of water, allowing an additional cultivation period in the dry season and/or in times of drought. Nevertheless, the nature of the highly weathered soils suggests that raised fields were not able to support large populations and their management required long fallow periods.

Comparison of two strategies for the treatment of acute myocardial infarction: In-hospital outcomes analysis

A strategy of pre-hospital reduced dose fibrinolytic administration coupled with urgent coronary intervention (PCI) for patients with STEMI (FAST-PCI) has been found to be superior to primary PCI (PPCI) alone. A coordinated STEMI system-of-care that includes FAST-PCI might offer better outcomes than pre-hospital diagnosis and STEMI team activation followed by PPCI alone. We compared the in-hospital outcomes for patients treated with the FAST-PCI approach with outcomes for patients treated with the PPCI approach during a pause in the FAST-PCI protocol. In-hospital data for 253 STEMI patients (03/2003–12/2009), treated with FAST-PCI protocol were compared to 124 patients (12/2009–08/2011), treated with PPCI strategy alone. In-hospital mortality was the primary endpoint. Stroke, major bleeding, and reinfarction during index hospitalization were secondary endpoints. Comparing the strategies used during the two time intervals, in-hospital mortality was significantly lower with FAST-PCI than with PPCI (2.77% vs. 10.48%, p = 0.0017). Rates of stroke, reinfarction and major bleeding were similar between the two groups. There was a lower frequency of pre- PCI TIMI 0 flow (no patency) seen in patients treated with FAST-PCI compared to the PPCI patients (26.7% vs. 62.7%, p<0.0001). Earlier infarct related artery patency in the FAST-PCI group had a favorable impact on the incidence of cardiogenic shock at hospital admission (FAST-PCI- 3.1% vs. PPCI- 20.9%, p<0.0001). The FAST-PCI strategy was associated with earlier infarct related artery patency and the lower incidence of cardiogenic shock on hospital arrival, as well as with reduced in-hospital mortality among STEMI patients.^

Characterizing Overall Response to Two-shot Vaccination for BVDV Type 2 in Angus Weanling Calves

Vaccination is a management strategy utilized to help reduce prevalence of bovine respiratory disease in feedlots. However, not all animals respond similarly to vaccinations. It is believed that an animal’s genetics control part of the ability to respond to a vaccination protocol. In order to evaluate the genetic control of a new trait such as response to vaccination, it is important to understand the non-genetic factors that affect an animal’s response to vaccination. The objective of this study was to characterize the non-genetic factors affecting overall response to a two-shot vaccination for bovine viral diarrhea virus type 2 (BVDV2) in Angus weanling calves.

Determination of sulfite and manganese in sediments from the K/T boundary

The solid-state-physics technique of electron spin resonance (ESR) has been employed in an exploratory study of marine limestones and impact-related deposits from Cretaceous-Tertiary (KT) boundary sites including Spain (Sopelana and Caravaca), New Jersey (Bass River), the U.S. Atlantic continental margin (Blake Nose, ODP Leg 171B/1049/A), and several locations in Belize and southern Mexico within -600 km of the Chicxulub crater. The ESR spectra of SO3(1-) (a radiation-induced point defect involving a sulfite ion substitutional for CO3(2-) which has trapped a positive charge) and Mn(2+) in calcite were singled out for analysis because they are unambiguously interpretable and relatively easy to record. ESR signal strengths of calcite-related SO3(1-) and Mn(2+) have been studied as functions of stratigraphic position in whole-rock samples across the KT boundary at Sopelana, Caravaca, and Blake Nose. At all three of these sites, anomalies in SO3(1-) and/or Mn(2+) intensities are noted at the KT boundary relative to the corresponding background levels in the rocks above and below. At Caravaca, the SO3(1-) background itself is found to be lower by a factor of 2.7 in the first 30,000 years of the Tertiary relative to its steady-state value in the last 15,000 years of the Cretaceous, indicating either an abrupt and quasi-permanent change in ocean chemistry (or temperature) or extinction of the marine biota primarily responsible for fixing sulfite in the late Cretaceous limestones. An exponential decrease in the Mn(2+) concentration per unit mass calcite, [Mn(2+)], as the KT boundary at Caravaca is approached from below (1/e characteristic length =1.4 cm) is interpreted as a result of post-impact leaching of the seafloor. Absolute ESR quantitative analyses of proximal impact deposits from Belize and southern Mexico group naturally into three distinct fields in a twodimensional [SO3(1-)]-versus-[Mn(2+)] scatter plot. These fields contain (I) limestone ejecta clasts, (II) accretionary lapilli, and (III) a variety of SO3(1-) -depleted/Mn(2+) enriched impact deposits. Data for the investigated non-impact-related Cretaceous and Tertiary marine limestones (Spain and Blake Nose) fall outside of these three fields. With reference to thes enon-impact deposits, fields I, II, and III can be respectively characterized as Mn(2+) -depleted, SO3(1-) -enhanced, and SO3(1-) -depleted. It is proposed that (1) field I represents calcites from the Yucatin Platform, and that the Mn(2+) -depleted signature can be used as an indicator of primary Chicxulub ejecta in deep marine environments and (2) field II represents calcites that include a component formed in the vapor plume, either from condensation in the presence of CO2/SO3(1-) -rich vapors, or reactions between CaO and CO2/SO3 rich vapors, and that this SO3(1-) -enhanced signature can be used as an indicator of impact vapor plume deposits. Given these two propositions, the ESR data for the Blake Nose deposits are ascribed to the presence of basal coarse calcitic Chicxulub ejecta clasts, while the finer components that are increasingly represented toward the top are interpreted to contain high- SO3(1-) calcite from the vapor plume. The apparently-undisturbed Bass River deposit may contain even higher concentrations of vapor-plume calcite. None of the three components included in field III appear to be represented at distal, deep marine KT-boundary sites; this field may include several types of impact-related deposits of diverse origins and diagenetic histories.

Description of sampling sites in Northern Russia

The youngest ice marginal zone between the White Sea and the Ural mountains is the W-E trending belt of moraines called the Varsh-Indiga-Markhida-Harbei-Halmer-Sopkay, here called the Markhida line. Glacial elements show that it was deposited by the Kara Ice Sheet, and in the west, by the Barents Ice Sheet. The Markhida moraine overlies Eemian marine sediments, and is therefore of Weichselian age. Distal to the moraine are Eemian marine sediments and three Palaeolithic sites with many C-14 dates in the range 16-37 ka not covered by till, proving that it represents the maximum ice sheet extension during the Weichselian. The Late Weichselian ice limit of M. G. Grosswald is about 400 km (near the Urals more than 700 km) too far south. Shorelines of ice dammed Lake Komi, probably dammed by the ice sheet ending at the Markhida line, predate 37 ka. We conclude that the Markhida line is of Middle/Early Weichselian age, implying that no ice sheet reached this part of Northern Russia during the Late Weichselian. This age is supported by a series of C-14 and OSL dates inside the Markhida line all of >45 ka. Two moraine loops protrude south of the Markhida line; the Laya-Adzva and Rogavaya moraines. These moraines are covered by Lake Komi sediments, and many C-14 dates on mammoth bones inside the moraines are 26-37 ka. The morphology indicates that the moraines are of Weichselian age, but a Saalian age cannot be excluded. No post-glacial emerged marine shorelines are found along the Barents Sea coast north of the Markhida line.

Enhancement of photosynthetic carbon assimilation efficiency by phytoplankton in the future coastal ocean

A mesocosm experiment was conducted to evaluate the effects of future climate conditions on photosynthesis and productivity of coastal phytoplankton. Natural phytoplankton assemblages were incubated in field mesocosms under the ambient condition (present condition: ca. 400 ppmv CO2 and ambient temp.), and two future climate conditions (acidification condition: ca. 900 ppmv CO2 and ambient temp.; greenhouse condition: ca. 900 ppmv CO2 and 3 °C warmer than ambient). Photosynthetic parameters of steady-state light responses curves (LCs; measured by PAM fluorometer) and photosynthesis-irradiance curves (P-I curves; estimated by in situ incorporation of 14C) were compared to three conditions during the experiment period. Under acidification, electron transport efficiency (alpha LC) and photosynthetic 14C assimilation efficiency (alpha) were 10% higher than those of the present condition, but maximum rates of relative electron transport (rETRm,LC) and photosynthetic 14C assimilation (PBmax) were lower than the present condition by about 19% and 7%, respectively. In addition, rETRm,LC and alpha LC were not significantly different between and greenhouse conditions, but PBmax and alpha of greenhouse conditions were higher than those of the present condition by about 9% and 30%, respectively. In particular, the greenhouse condition has drastically higher PBmax and alpha than the present condition more than 60% during the post-bloom period. According to these results, two future ocean conditions have major positive effects on the photosynthesis in terms of energy utilization efficiency for organic carbon fixation through the inorganic carbon assimilation. Despite phytoplankton taking an advantage on photosynthesis, primary production of phytoplankton was not stimulated by future conditions. In particular, biomass of phytoplankton was depressed under both acidification and greenhouse conditions after the the pre-bloom period, and more research is required to suggest that some factors such as grazing activity could be important for regulating phytoplankton bloom in the future ocean.