Nutrient Conditions Research Articles

Tectorotularia hexagona (Polychaeta, Serpulidae) co-existing with Rhynchostreon suborbiculatum (Bivalvia, Gryphaeidae) in a river-dominated marginal marine environment of Cenomanian–Turonian age in Slovakia: An example of Late Cretaceous amensalism or competition?

The mutual relationship and co-occurrence of the tube-dwelling polychaete Tectorotularia hexagona and the oyster Rhynchostreon suborbiculatum in an instance of fluctuating marginal marine environment, represented by the Orlové Sandstone in the Western Carpathians (Pieniny Klippen Belt), is recorded. As a consequence of climate change during the Cenomanian and early Turonian, changes over time in key ecological factors (e.g., decrease in freshwater inflow and increase in temperature and water mass salinity) led to critical trophic alterations in the marginal marine system studied. The environmental change from polyhaline/eutrophic to euhaline/lower nutrient conditions led to a reduction in the pioneer palaeopopulation of R. suborbiculatum, and Tectorotularia hexagona and other stenohaline marine recliners took over the ecological niches originally occupied by the oyster. In this respect, the relationship between T. hexagona and R. suborbiculatum may be considered a Late Cretaceous example of competition between two groups of sessile-benthic recliners. In the present work, two different tube morphologies of T. hexagona from Hôrka are described, and the tube microstructure of this species is documented for the first time. In addition, the material from Hôrka is compared with that from the type locality, Essen in Germany, and ‘Hamulus’ hexagonus and Tectorotularia ‘westfalica’ are considered to constitute one and the same species. The wider ecological and systematic context of this serpulid species is discussed as well.

Discriminating bloom-forming cyanobacteria using lab-based hyperspectral imagery and machine learning: Validation with toxic species under environmental ranges

Cyanobacteria are major contributors to algal blooms in inland waters, threatening ecosystem function and water uses, especially when toxin-producing strains dominate. Here, we examine 140 hyperspectral (HS) images of five representatives of the widespread, potentially toxin-producing and bloom-forming genera Microcystis, Planktothrix, Aphanizomenon, Chrysosporum and Dolichospermum, to determine the potential of utilizing visible and near-infrared (VIS/NIR) reflectance for their discrimination. Cultures were grown under various light and nutrient conditions to induce a wide range of pigment and spectral variability, mimicking variations potentially found in natural environments. Importantly, we assumed a simplified scenario where all spectral variability was derived from cyanobacteria. Throughout the cyanobacterial life cycle, multiple HS images were acquired along with extractions of chlorophyll a and phycocyanin. Images were calibrated and average spectra from the region of interest were extracted using k-means algorithm. The spectral data were pre-processed with seven methods for subsequent integration into Random Forest models, whose performances were evaluated with different metrics on the training, validation and testing sets. Successful classification rates close to 90 % were achieved using either the first or second derivative along with spectral smoothing, identifying important wavelengths in both the VIS and NIR. Microcystis and Chrysosporum were the genera achieving the highest accuracy (>95 %), followed by Planktothrix (79 %), and finally Dolichospermum and Aphanizomenon (>50 %). The potential of HS imagery to discriminate among toxic cyanobacteria is discussed in the context of advanced monitoring, aiming to enhance remote sensing capabilities and risk predictions for water bodies affected by cyanobacterial harmful algal blooms.

Impacts of AlaAT3 transgenic poplar on rhizosphere soil chemical properties, enzyme activity, bacterial community, and metabolites under two nitrogen conditions

ABSTRACT Poplar stands as one of the primary afforestation trees globally. We successfully generated transgenic poplar trees characterized by enhanced biomass under identical nutrient conditions, through the overexpression of the pivotal nitrogen assimilation gene, pxAlaAT3. An environmental risk assessment was conducted for investigate the potential changes in rhizosphere soil associated with these overexpressing lines (OL). The results show that acid phosphatase activity was significantly altered under ammonium in OL compared to the wild-type control (WT), and a similar difference was observed for protease under nitrate. 16SrDNA sequencing indicated no significant divergence in rhizosphere soil microbial community diversity between WT and OL. Metabolomics analysis revealed that the OL caused minimal alterations in the metabolites of the rhizosphere soil, posing no potential harm to the environment. With these findings in mind, we anticipate that overexpressed plants will not adversely impact the surrounding soil environment.

Dynamic adaptation of phytoplankton vertical migration to changing grazing and nutrient conditions

Despite the ubiquitous occurrence of vertical migration of phytoplankton its quantitative significance remains poorly known. We eliminated a dense Daphnia population in a pond by introducing whitefish fingerlings, and assessed the effects on the vertical migration of dominating motile phytoplankton. At the highest abundance of Daphnia, cryptophytes reduced grazing losses by staying in the hypolimnion day and night, but Mallomonas species armoured by silica bristles remained in the epilimnion. After the fish introduction, phytoplankton was released from Daphnia grazing pressure, allowing cryptophytes to occur in the epilimnion also at noon. At the same time, increased phytoplankton biomass exacerbated the nutrient depletion. Cryptophytes compensated for that by migrating into the anoxic hypolimnion, whereupon their growth rates increased. The collapse of Daphnia was also associated with a temporary increase in nutrient regeneration by enzyme activities and decreases in total nutrient concentration and bacterial biomass in the whole water column. Our results show that cryptophytes can dynamically modify their vertical migration to balance between the exploitation of various nutrient resources and the risk of becoming eaten. Hypolimnetic nutrient resources can be quantitatively more important for phytoplankton than previously assumed.

Optimisation of Saccharomyces cerevisiae BRYC 501 ascospore formation and recovery for heat inactivation experiments

Why was the work done: Ascospores from Saccharomyces cerevisiae BRYC 501 are useful biological indicators for validating the pasteurisation of beer. Ascospores are formed by yeast in response to low nutrient conditions and are characterised by greater resistance to stressors including desiccation, freezing and, during pasteurisation, high temperatures. Accordingly, the sporulation temperature of yeast ascospores, their shelf life, heat resistance and recovery need to be investigated. How was the work done: The role of temperature in sporulation was investigated at 25, 27 and 30°C by measurement of the rate of sporulation over ten days. Shelf life of ascospores was established in deionised water at 1-5°C over 120 days by the measurement of heat resistance in McIlvaine's buffer. The recovery conditions of ascospores were determined using six agars after heat inactivation experiments in McIlvaine's buffer, alcohol free and lager beer. What are the main findings: The sporulation rate of S. cerevisiae ascospores was highest at 27°C, and lowest at 30°C. Heat resistance of ascospores formed at 30°C was low with a D60 value of <3 minutes, whereas at 25 and 27°C the D60 was 8.8 and 9.2 minutes. The shelf life of ascospores was 60 days after an initial 14 days of maturation at both 25 and 27°C. The variation of heat resistance for ascospores grown at 25°C was shown to be lower than those grown at 27°C. Use of yeast extract-peptone-glucose agar to recover heat injured cells was > 200% higher than with the control agar. Why is the work important: Sporulation should be performed at 25°C to produce the maximum number of spores for heat inactivation experiments. Spores can be maintained for 60 days, enabling several experiments to be performed. After heat inactivation experiments, ascospores should be recovered on YEPG agar for 10 days at 27°C

Does soil nutrient heterogeneity affect the competition and adaptation of Vernicia montana?

Plants respond to variations in soil nutrient distribution and the presence of neighboring plants, yet consensus is lacking on the impact of naturally heterogeneous soil nutrient environments on plant competition. This study employs pot experiments to simulate homogeneous and heterogeneous forest soil nutrient environments and designs neighbor competition experiments for Vernicia montana, Cunninghamia lanceolata, and Phoebe bournei, to assess the impact of neighbor competition on these seedlings from the perspective of root morphology and foraging ability in different nutrient environments. The results reveal that when faced with heterogeneous nutrient environments or competitive settings, V. montana primarily utilized morphological and physiological plasticity, such as increasing root morphological indicators and concentrating more nutrients in the underground part, to enhance its adaptability and achieve competitive advantages. C. lanceolata and P. bournei mainly depended on physiological plasticity, like increased root nutrient concentration, when they were co-planted with other species. Compared to interspecific competition, intraspecific competition had a significant inhibitory effect on V. montana, especially under heterogeneous nutrient conditions, with notable reductions in root morphological indicators and root N, P concentration in mono-culture. Nutrient heterogeneity also altered the effects of interspecific competition on C. lanceolata and P. bournei. When mixed-planted with C. lanceolata or P. bournei, V. montana exhibited stronger competitive ability in both nutrient environments. The effects of mixed planting on C. lanceolata and P. bournei were significantly different between the two nutrient environments, in that homogeneous nutrient environment favored the growth and nutrient absorption of C. lanceolata, while heterogeneous nutrients generally benefited P. bournei. Most research on seedling competition is conducted under homogenous nutrient environments, and the results may not accurately reflect in-situ competition, potentially obscuring the scientific implementation of forestry management. Taking into account competition under unevenly distributed soil resources will help in optimizing tree species selection and cultivation methods, thereby improving the adaptability and ecological benefits of forests.

Morphological and physiological responses of the cosmopolitan marine diatom Thalassiosira rotula to acidification

We investigated the effect of decreased pH on the morphology and nutrient physiology of the cosmopolitan marine diatom Thalassiosira rotula (CCMP3362) by acclimating unialgal cultures to two different CO2 gas concentrations under optimal light, temperature and nutrient conditions. At lower pH (higher CO2), T. rotula exhibited a reduction in cell diameter (7%), surface area (13%), and volume (20%), and an increase in surface area-to-volume ratio (7%). All measures of silicification in T. rotula, i.e., silica (SiO2) quota, rate of silicic acid (Si(OH)4) utilization, and elemental ratios of SiO2:C and SiO2:N, remained unchanged. Similarly, carbon (C) and nitrogen (N) quotas, ratios and utilization rates were mostly unaffected by pH. In contrast, the utilization rate of nitrate (ρNO3) was significantly lower at decreased pH when the rate was normalized by cell number instead of by cell volume. The changes in cell morphology found in this study under low pH were likely not large enough to significantly impact physiological processes and the role of this species in marine food webs and biogeochemical cycles. It is possible that the interactive effects of pH, temperature, light, and nutrient availability characteristic of different regions of the world ocean could result in stronger physiological and morphological responses in this widespread diatom. However, under constant optimal growth conditions, T. rotula was only mildly affected by changes in pH, and in particular the physiology and elemental stoichiometry of C, N and Si were not sensitive to acidification.

Human Plasma-Like Medium Activates Immune Elements in Glioma

INTRODUCTION: Glioma Surgically eXplanted Organoids (SXOs) are ex vivo three-dimensional models demonstrated to recapitulate the parental tumor microenvironment (TME). Human plasma-like medium (HPLM), a culture medium that mirrors physiologic nutrient conditions, has been found in other cancer subtypes to induce transcriptional differences in T-lymphocyte activation. We hypothesized that SXO culture in HPLM would maintain immune cell populations and induce physiologically relevant immune-modulatory pathways. METHODS: SXOs were established and cultured in conventional Glioma Organoid Complete medium (GOC) and transitioned to HPLM for 24 or 120 hours. Formalin-fixed paraffin-embedded samples were stained for tumor cells (GFAP), immune cells (CD45), and nucleic acid (SYTO-13) and analyzed using the Nanostring GeoMx™ Digital Spatial Profiler for spatial transcriptomics. Gene expression clustering profiles were conducted using GSEA and CIBERSORT. The gene expression differences of SXOs cultured in HPLM for 24 and 120 hours compared to those in GOC were analyzed by unpaired t-tests. RESULTS: Glioma SXOs retain diverse cell populations, with 20-30% of cells belonging to the TME. CD4+ T-cells were the most common (9.7 ± 1.0), followed by CD8+ T-cells (4.4 ± 0.6) and macrophages (3.1 ± 1.2). Following the transition to HPLM for 24 or 120 hours, there were no significant differences in tumor, immune, and stromal cell fractions. HPLM culture upregulated the immune response in SXOs, encompassing enhanced cytokine secretion, as well as improved adaptive and innate immune pathways. We particularly highlighted CD4+ T-cell activation (CD69+), Treg, and TGF-β signaling pathways as significantly increased. CONCLUSIONS: Here, we present that the culture of SXOs in physiologically similar media retains the heterogeneous TME and activates innate and adaptive immune signaling pathways. Culture in HPLM enables investigation of the glioma immune microenvironment, expanding the biological relevance of experimental results from in vitro SXO models.

Greater capacity for division of labor brings greater benefits to an invasive clonal plant than its native partners

The majority of problematic invasive plants exhibit clonal growth, and the clonal traits are widely recognized as critical determinants contributing to their invasiveness. Division of labor represents a crucial advantage associated with clonal growth; therefore, it is reasonable to anticipate that this particular trait may confer a growth advantage upon invasive clonal species in comparison to native ones. To test this hypothesis, we conducted a comparative experiment in the greenhouse involving the highly invasive plant Alternanthera philoxeroides, its native congener A. sessilis, and its native co-occurring Jussiaea repens. We grew clonal fragments of these three stoloniferous plants under negative spatial covariance of light and soil nutrients while either preventing or allowing division of labor through severing or keeping intact their stolon connections. The results showed that stolon connection significantly increased the root mass to shoot mass ratio of basal ramets under low light and high soil nutrient conditions, but greatly decreased it in the apical ramets under high light and low soil nutrient conditions, suggesting induced division of labor in all studied plants. Moreover, the invasive plant A. philoxeroides demonstrated a greater capacity for division of labor than its co-genus and co-occurring native partners, which conferred it with greater growth performance at the whole clonal fragment level. Given that the commonly observed competitive superiority of invasive clonal plants over co-occurring native species due to their high intrinsic growth rates, these findings suggest that the division of labor may confer a competitive advantage upon some invasive clonal plants, thereby facilitating their successful invasion in heterogeneous habitats characterized by a strong negative correlation between light availability and soil nutrient levels.

Carbon sequestration potential of biochar in soil from the perspective of organic carbon structural modification

Application of biochar derived from biomass resources in soil is an encouraging method to decelerate global warming via carbon sequestration. While there has been extensive research on the priming effect of biochar on the mineralization of native soil organic carbon (nSOC), its correlation with soil organic carbon (SOC) structural variations remains poorly understood. A series of incubation experiments with soils amended with biochar prepared at 300 °C, 450 °C, and 600 °C were performed to explore the shifts in nSOC structure and reveal the interconnections among soil properties, SOC structural variation, and the priming effect induced by biochar. Biochar exhibited an “inhibitory concentration” on soil microorganisms, where 1 % biochar enhanced bacterial and fungal richness and diversity, while 3 % biochar reduced bacterial richness and diversity although soil nutrient conditions were improved. Biochar-treated soil had higher humic acid- and humin-like fractions (HAL and HML) but lower fulvic acid-like components (FAL). Moreover, bulk SOC, FAL, HAL, and HML of soils with 300 °C and 450 °C biochar contained more aromatic-type species (e.g., lignin). Such changes were related to improvement of the soil nutrient condition and shifts in microbial community structures, in addition to the “accumulation of biochar-derived OC in the nSOC pool”. Stable carbon isotopes indicated that the inhibitory effect on SOC mineralization (−94.5 to −8.0 mg CO2-C kg−1 soil) induced by biochar was significantly explained by the change in FAL and HAL contents. The findings improved the understanding of the carbon sequestration potential of biochar in soils for SOC structural modification.

Glucosylglycerol and proline reverse the effects of glucose on Rhodosporidium toruloides lifespan.

Rhodosporidium toruloides is a novel cell factory used to synthesis carotenoids, biosurfactants, and biofuel feedstocks. However, research on R. toruloides has generally centred on the manufacture of biochemicals, while analyses of its longevity have received scant attention. Understanding of R. toruloides longevity under different nutrient conditions could help to improve its biotechnological significance and metabolite production. Glucosylglycerol (GG) and proline are osmoprotectants that could revert the harmful effects of environmental stress. This study examined how GG and proline affect R. toruloides strain longevity under glucose nutrimental stress. Herein, we provide evidence that GG and proline enhance cell performance and viability. These compatible solutes neutralises the pro-ageing effects of high glucose (10% glucose) on the yeast cell and reverse its cellular stress. GG exhibits the greatest impact on lifespan extension at 100mM, whereas proline exerts effect at 2mM. Our data reveal that these compounds significantly affect the culture medium osmolarity. Moreso, GG and proline decreased ROS production and mitohormetic lifespan regulation, respectively. The data indicates that these solutes (proline and GG) support the longevity of R. toruloides at a pro-ageing high glucose culture condition.

Fluctuations in Radiation Use Efficiency Throughout the Growth Cycle in Diploid Potato Crop

The capture of incoming solar radiation under unlimited light, water, and nutrient conditions by plant canopies and converting it into biomass has been described as radiation use efficiency (RUE). RUE has been computed as a function of biomass accumulation and intercepted photosynthetically active radiation without considering the loss of photoassimilates due to respiratory processes. This study evaluated the RUE in diploid potato crop (Solanum phureja Juz. et Buk.) across six experiments in Colombia. Total biomass was measured during the crop season from the early vegetative stage through maturity. However, this proposal involves not only the total biomass accumulated concerning the amount of photosynthetically active radiation intercepted but also took into account the losses by respiration, following Thornley respiration approach. This research demonstrates that the RUE is not a constant value as the respiration process leads to RUE values being variable in a non-linear way over time. The daily RUE simulation, conducted through an interpolation process, revealed significant variation from emergence to the end of the cycle. This indicates an error in assuming a constant RUE throughout the entire growth period, particularly in assessing its physiological impact across the entire growth and development crop cycle.

Driving mechanism of different nutrient conditions on microbial mediated nitrate reduction in magnetite-present river infiltration zone

Significant research is focused on the ability of riparian zones to reduce groundwater nitrate contamination. Owing to the extremely high redox activity of nitrate, naturally existing electron donors, such as organic matter and iron minerals, are crucial in facilitating nitrate reduction in the riparian zone. Here, we examined the coexistence of magnetite, an iron mineral, and nitrate, a frequently observed coexisting system in sediments, to investigate nitrate reduction features at various C/N ratios and evaluate the response of microbial communities to these settings. Additionally, we aimed to use this information as a foundation for examining the effect of nutritional conditions on the nitrate reduction process in magnetite-present environments. These results emphasise the significance of organic matter in enabling dissimilatory nitrate reduction to ammonium (DNRA) and enhancing the connection between nitrate reduction and iron in sedimentary environments. In the later phases of nitrate reduction, nitrogen fixation was the prevailing process in low-carbon environments, whereas high-carbon environments tended to facilitate the breakdown of organic nitrogen. High-throughput sequencing analysis revealed a robust association between C/N ratios and alterations in microbial community composition, providing insights into notable modifications in essential functioning microorganisms. The nitrogen-fixing bacterium Ralstonia is more abundant in ecosystems with scarce organic matter. In contrast, in settings rich in organic matter, microorganisms, such as Acinetobacter and Clostridia, which may produce ammonia, play crucial roles. Moreover, the population of iron bacteria grows in such an environment. Hence, this study proposes that C/N ratios can influence Fe(II)/Fe(III) conversions and simultaneously affect the process of nitrate reduction by shaping the composition of specific microbial communities.

Fertilizer regime and cultivar affect barley growth and rhizobiome composition

To combat climate change and environmental pollution, agriculture must reduce mineral fertilizer use and adopt sustainable, low-input, and organic practices. In such systems, plants depend on the soil microbiome for nutrient acquisition and growth. Thus, enhancing plant-microbiome interactions is vital for maintaining or even increasing production sustainably. Modern plant breeding has led to barley cultivars that yield high under substantial mineral fertilizer application. However, this selective breeding may have diminished traits essential for plant-microbiome interactions, making these cultivars less suitable for sustainable agriculture. In contrast, older cultivars might have preserved traits from their common wild ancestor, enabling them to prosper under low nutrient conditions. This study evaluated four modern elite barley cultivars and three older (pre-1980). Grown under various fertilizer regimes, we assessed their root microbiome using 16S rRNA amplicon sequencing. Our objectives were: i) to determine the impact of nutrient availability on plant nutrient uptake and biomass production, and ii) to understand how the time since domestication (domestication age), individual cultivar, and fertilizer regime influence the root microbiome. We found that without fertilizer, old cultivars had superior biomass production and higher leaf concentrations of nitrogen, potassium, sulfur, iron, zinc, and copper than modern cultivars. This indicates that older barley cultivars may have retained their wild ancestors' capability to synergize with the soil microbiome, enhancing nutrient acquisition in low-input systems. Notably, the diversity of the rhizo-microbiome was not significantly affected by domestication age but varied with individual cultivar and fertilizer treatment.

Abstract 6606: HK2 is conditionally essential for growth in human cancer cells

Abstract The genetic drivers of cell growth can depend on an interplay of intrinsic factors and environmental context. However, there is often little consideration into how nutrient availability affects the genetic dependencies of proliferating human cells. Moreover, efforts such as DepMap have catalogued cell fitness genes using in vitro CRISPR screens across hundreds of cancer cell lines cultured in traditional media, which poorly replicate nutrient conditions found in the body. We recently tested the hypothesis that the nutrient environment can affect gene essentiality in human cells. By performing CRISPR screens with blood cancer cell lines, we identified sets of genes differentially required for cells cultured in traditional (e.g., RPMI) versus Human Plasma-Like Medium (HPLM). Among the strongest HPLM-essential hits was HK2, which encodes for the glycolytic enzyme hexokinase 2. Notably, most cancer cell lines co-express two hexokinases (HK1; HK2) that can each catalyze the phosphorylation of glucose to glucose-6-phosphate. Interestingly, while these two HKs share high degrees of structural and biochemical similarity, only HK2 deletion had differentially stronger effects in HPLM. Further, both HK1 and HK2 contain an N-terminal region that permits association with the outer mitochondrial membrane (OMM). To investigate the conditional phenotype for loss of HK2, we generated HK2-knockout K562 leukemia cells, which displayed HK1 expression levels comparable to those measured in control cells. We also confirmed that our HK2-knockout cells showed a stronger relative growth defect in HPLM versus RPMI. Importantly, the expression of HK2 cDNA normalized this defect. By contrast, the expression of kinase-dead HK2 did not provide any rescue effects, suggesting that HK activity is required for the conditionally essential role of HK2. We also engineered a cytosol-restricted HK2 by truncating its N-terminal OMM binding region. When we expressed this cDNA in our HK2-knockout cells, we observed complete rescue of the relative growth defect, suggesting that subcellular HK2 activity contributes to conditional HK2 essentiality. We are now purifying mitochondria to ask how the subcellular distributions of HK1 and HK2 vary with nutrient conditions. In addition, we are also comparing the tracing patterns of 13C glucose in HK2-knockout and control cells to ask how HK2 deletion affects glucose utilization. Finally, given that we normalized glucose levels across our screening conditions, we are applying a systematic approach to determine the gene-nutrient interaction that underlies the conditional phenotype for HK2 deletion. Collectively, our data suggest that HK2 serves a non-redundant and conditionally essential role. We expect to generate novel insights into how HK2 supports human cell growth and why this role can further depend on nutrient availability. Citation Format: Kimberly S. Huggler, Kyle M. Flickinger, Jason R. Cantor. HK2 is conditionally essential for growth in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6606.

Abstract 1800: Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells

Abstract The genes that are required for the growth of mammalian cells can depend on an interplay of cell-intrinsic factors and environmental context. However, there is often little investigation into how nutrient availability impacts gene essentiality. Moreover, efforts such as DepMap have catalogued genes that contribute to cancer cell fitness using in vitro CRISPR screens in hundreds of cancer cell lines cultured in traditional media that poorly recapitulate the nutrient availability of human blood. Previously, we developed Human Plasma-Like Medium (HPLM), a physiologic medium designed to better model the nutrient availability of human blood. We recently tested the hypothesis that nutrient availability influences gene essentiality. By performing paired CRISPR-based screens in human blood cancer cells, we identified sets of genes that are differentially required to support cell growth in HPLM versus traditional media such as RPMI. Among the strongest scoring HPLM-essential genes was NAD kinase (NADK), which encodes an enzyme critical for the generation of cytosolic NADPH. NADK has been previously suggested as an anti-cancer therapeutic target, given the central importance of NADPH for enabling proliferative metabolism through macromolecule synthesis and ROS management. Interestingly, NADK has been annotated as an essential gene in only 1% of the cell lines included in DepMap, suggesting that the essentiality of NADK may have been largely masked by the nutrient conditions used to generate the DepMap. To investigate the conditional CRISPR phenotype for NADK, we engineered NADK-knockout cells and found that they showed a 50% stronger growth defect versus control cells in HPLM relative to RPMI. Next, we used localization and activity studies to confirm that cytosolic NAD+ kinase activity is required for the conditionally essential role of NADK. We then employed systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene-nutrient interaction and to gain insights into the conditionally essential role of NADK. Through these studies, we traced the cause of NADK essentiality to an inhibition of folate-dependent nucleotide synthesis linked to differential folic acid availability. Finally, we examined the pre-translational potential of NADK as an anti-cancer therapeutic target by demonstrating that the reported NADK inhibitor thionicotinamide largely phenocopies NADK-knockout in a folic-acid dependent manner in the K562 cell line. Collectively, our work improves understanding of how NADK contributes to human cell growth and reveals how the relative importance of cytosolic NAD+ kinase activity can depend on nutrient availability. Citation Format: Kyle M. Flickinger, Kimberly S. Huggler, Jason R. Cantor. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1800.

Environmental Factors Influencing Annual Changes in Bycatch per Unit Effort of Delphinus delphis around Their Main Hotspot in Korean Waters

In this study, the characteristics of temporal changes in bycatch per unit effort (BPUE) as an index of the relative density of the common dolphin around their widest hotspot in the East Sea/Sea of Japan were examined from 2011 to 2021. BPUE rapidly increased from 2017 to 2019. The annual changes in BPUE were due to changes during March–April, which is the most abundant season for common dolphins. The annual relationship between BPUE and four variables (Pacific herring, common squid catches, chlorophyll-a concentration, and sea surface temperature) around their main hotspot for March–April was investigated using generalized linear models (GLMs) for gamma distribution. A stepwise Bayesian information criterion for the gamma GLM yielded significant retention of two variables, Pacific herring catch and chlorophyll-a concentration, over the study period, indicating that the rapid increase of the relative density of the common dolphin in the early spring during 2017–2019 could be due to the increase of their prey abundance caused by higher primary productivity. Therefore, ecosystem productivity altered by nutrient conditions could be a key biological process that enhances habitat use of cetaceans in highly productive seasons and regions.

Abstract 375: Lipogenic phenotype is an adaptation to nutrient stress in the tumor microenvironment

Abstract The nutrient and oxygen poor microenvironment combined with acidosis arising from fermentative metabolism imparts strong selection pressure on cancer cells that promotes evolution of more aggressive cancer types. Poor nutrient availability in the tumor microenvironment forces cancer cells to adapt to alternate routes of metabolism, nutrient scavenging and autophagy leading to a shift in their metabolic phenotype. These adaptations promote evolution of invasive and aggressive phenotypes. To test this hypothesis and to characterize the evolution of cancer cells under nutrient stress, we generated nutrient stress adapted cells from MCF7-GFP cells by subjecting them to multiple cycles of nutrient starvation and repletion. Distinct clones that outgrew were selected as nutrient stress adapted cells (NSA). Here we show that MCF7-NSA cells that are adapted to grow in low nutrient conditions exhibited robust accumulation of lipid droplets compared to parental MCF7-GFP cells as shown by confocal microscopy after neutral lipid staining as well as immunofluorescence staining for PLIN2 (Perilipin 2), a lipid droplet coat protein. In addition, MCF7-NSA cells showed elevated expression of the nuclear receptor PPARγ and the adipokine FABP4 indicating an adipogenic/lipogenic signature. Strikingly, these nutrient stress adapted cells were capable of nutrient scavenging by macropinocytosis compared to control cells even under nutrient replete conditions. Macropinocytosis was assessed by DQ-BSA processing and TMR-Dextran uptake. NHE inhibitor EIPA (ethyl isopropyl amiloride), that blocks macropinocytosis, but not receptor mediated endocytosis confirmed dextran uptake through macropinocytosis. Similarly, macropinocytosis inhibitor KH7 blocked dextran uptake in these adapted cells. Furthermore, MCF7-NSA cells showed high expression of TFEB and TFE3, master regulators of autophagy and lysosomogenesis suggesting that the necrotic cell debris scavenged through macropinocytosis and processed through autolysosome machinery might be contributing to de novo lipogenesis. Although control and adapted cells exhibited similar growth kinetics in vitro, when implanted in mammary fat pads (MFP) of NSG mice, NSA cells formed significantly smaller and slow growing primary tumors. Interestingly, 80% of these mice showed metastasis to other organs (lung, liver, kidney) whereas control mice did not develop metastasis as parental MCF7 is not an aggressive cell line in xenograft models. MCF7-NSA cells were more migratory in vitro in wound healing assays and were capable of developing lung metastases in a mouse model where primary tumor from mammary fat pad was resected to allow for metastases to develop. Taken together, our data suggest that lipogenic phenotype confers survival advantage in the harsh tumor microenvironment and selects for more invasive and aggressive phenotypes. Citation Format: Crystal Griffith, Dominique Abrahams, Antonio Ortiz, Alexandra Tassielli, Joseph Johnson, Robert Gatenby, Smitha Pillai. Lipogenic phenotype is an adaptation to nutrient stress in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 375.

The acyl-CoA synthetase TgACS1 allows neutral lipid metabolism and extracellular motility in Toxoplasma gondii through relocation via its peroxisomal targeting sequence (PTS) under low nutrient conditions.

Apicomplexa parasites cause major diseases such as toxoplasmosis and malaria that have major health and economic burdens. These unicellular pathogens are obligate intracellular parasites that heavily depend on lipid metabolism for the survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs) obtained from both de novo synthesis and scavenging from the host. The constant flux of scavenged FA needs to be channeled toward parasite lipid storage, and these FA storages are timely mobilized during parasite division. In eukaryotes, the utilization of FA relies on their obligate metabolic activation mediated by acyl-co-enzyme A (CoA) synthases (ACSs), which catalyze the thioesterification of FA to a CoA. Besides the essential functions of FA for parasite survival, the presence and roles of ACS are yet to be determined in Apicomplexa. Here, we identified TgACS1 as a Toxoplasma gondii cytosolic ACS that is involved in FA mobilization in the parasite specifically during low host nutrient conditions, especially in extracellular stages where it adopts a different localization. Heterologous complementation of yeast ACS mutants confirmed TgACS1 as being an Acyl-CoA synthetase of the bubble gum family that is most likely involved in β-oxidation processes. We further demonstrate that TgACS1 is critical for gliding motility of extracellular parasite facing low nutrient conditions, by relocating to peroxisomal-like area.IMPORTANCEToxoplasma gondii, causing human toxoplasmosis, is an Apicomplexa parasite and model within this phylum that hosts major infectious agents, such as Plasmodium spp., responsible for malaria. The diseases caused by apicomplexans are responsible for major social and economic burdens affecting hundreds of millions of people, like toxoplasmosis chronically present in about one-third of the world's population. Lack of efficient vaccines, rapid emergence of resistance to existing treatments, and toxic side effects of current treatments all argue for the urgent need to develop new therapeutic tools to combat these diseases. Understanding the key metabolic pathways sustaining host-intracellular parasite interactions is pivotal to develop new efficient ways to kill these parasites. Current consensus supports parasite lipid synthesis and trafficking as pertinent target for novel treatments. Many processes of this essential lipid metabolism in the parasite are not fully understood. The capacity for the parasites to sense and metabolically adapt to the host physiological conditions has only recently been unraveled. Our results clearly indicate the role of acyl-co-enzyme A (CoA) synthetases for the essential metabolic activation of fatty acid (FA) used to maintain parasite propagation and survival. The significance of our research is (i) the identification of seven of these enzymes that localize at different cellular areas in T. gondii parasites; (ii) using lipidomic approaches, we show that TgACS1 mobilizes FA under low host nutrient content; (iii) yeast complementation showed that acyl-CoA synthase 1 (ACS1) is an ACS that is likely involved in peroxisomal β-oxidation; (iv) the importance of the peroxisomal targeting sequence for correct localization of TgACS1 to a peroxisomal-like compartment in extracellular parasites; and lastly, (v) that TgACS1 has a crucial role in energy production and extracellular parasite motility.

METTL16 deficiency attenuates apoptosis through translational control of extrinsic death receptor during nutrient deprivation

METTL16 is a well-characterized m6A methyltransferase that has been reported to contribute to tumorigenesis in various types of cancer. However, the effect of METTL16 on tumor progression under restricted nutrient conditions, which commonly occur in tumor microenvironment, has yet to be elucidated. Herein, our study initially reported the inhibitory effect of METTL16 depletion on apoptosis under amino acid starvation conditions. Mechanistically, we determined that the METTL16 knockdown represses the expression of extrinsic death receptors at both transcription and translation levels. Depletion of METTL16 prevented protein synthesis of GCN2, resulting in diminished ATF4 expression in a GCN2-eIF2α-dependent manner. Reduction of ATF4 further declined the expression of apoptotic receptor protein DR5. Meanwhile, METTL16 deficiency directly hampered protein synthesis of FADD and DR5, thereby impairing apoptosis and promoting cancer cell survival. Taken together, our study provides novel evidence for the involvement of METTL16 in regulating cancer progression, suggesting that METTL16 as a potential therapeutic target for cancer treatment.