Neutral Lipid Content Research Articles

Inhibiting the Cholesterol Storage Enzyme ACAT1/SOAT1 in Myelin Debris-Treated Microglial Cell Lines Activates the Gene Expression of Cholesterol Efflux Transporter ABCA1

Aging is the major risk factor for Alzheimer’s disease (AD). In the aged brain, myelin debris accumulates and is cleared by microglia. Phagocytosed myelin debris increases neutral lipid droplet content in microglia. Neutral lipids include cholesteryl esters (CE) and triacylglycerol (TAG). To examine the effects of myelin debris on neutral lipid content in microglia, we added myelin debris to human HMC3 and mouse N9 cells. The results obtained when using 3H-oleate as a precursor in intact cells reveal that myelin debris significantly increases the biosynthesis of CE but not TAG. Mass analyses have shown that myelin debris increases both CE and TAG. The increase in CE biosynthesis was abolished using inhibitors of the cholesterol storage enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1/SOAT1). ACAT1 inhibitors are promising drug candidates for AD treatment. In myelin debris-loaded microglia, treatment with two different ACAT1 inhibitors, K604 and F12511, increased the mRNA and protein content of ATP-binding cassette subfamily A1 (ABCA1), a protein that is located at the plasma membrane and which controls cellular cholesterol disposal. The effect of the ACAT1 inhibitor on ABCA1 was abolished by preincubating cells with the liver X receptor (LXR) antagonist GSK2033. We conclude that ACAT1 inhibitors prevent the accumulation of cholesterol and CE in myelin debris-treated microglia by activating ABCA1 gene expression via the LXR pathway.

Optimizing Nitrate and Tryptone to Enhance Growth and Triacylglycerol Accumulation in Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a unicellular diatom, is considered a potential feedstock for the production of biofuel and a promising producer for high-value products eicosapentaenoic acid and fucoxanthin. However, a high-efficient cultivating strategy to achieve commercial production of triacylglycerol (TAG) from the diatom is an urgent demand. In this study, we optimized the content and ratio of nitrate and tryptone in the medium to enhance biomass and TAG accumulation simultaneously. Growth with tryptone as the sole nitrogen gave rise to the lowest cell density but the highest TAG content in P. tricornutum relative to nitrate, nitrite, ammonium or urea cultures. In 500 μM NaNO3 cultures, the growth of P. tricornutum increased with the increasing concentration (from 294 to 7056 μM nitrogen) of supplemented tryptone, however supplementation of high tryptone (≥882 μM nitrogen) decreased the neutral lipid content. Elevating nitrogen concentration from 294 to 882 μM via tryptone addition in 250 μM nitrate culture increased cell densities from day 6 to 10 and neutral lipid content on day 10. In particular, supplementing 588 μM nitrogen of tryptone in the 250 μM nitrate culture gave rise to the highest neutral lipid content on days 8 and 10 (increased by 109% and 62% relative to 500 μM nitrate-sole) with a comparable growth to that in 500 μM nitratesole culture from day 2 to 8. In conclusion, we optimized nitrate/tryptone ratio and found that a suitable tryptone addition to a relatively low nitrate culture was favourable to the biomass and TAG accumulation simultaneously in P. tricornutum.

Administration of Essential Phospholipids Prevents Drosophila Melanogaster Oocytes from Responding to Change in Gravity.

The aim of this study was to prevent initial changes in Drosophila melanogaster oocytes under simulated weightlessness and hypergravity at the 2 g level. Phospholipids with polyunsaturated fatty acids in the tail groups (essential phospholipids) at a concentration of 500 mg/kg of nutrient medium were used as a protective agent. Cell stiffness was determined using atomic force microscopy, the change in the oocytes' area was assessed as a mark of deformation, and the contents of cholesterol and neutral lipids were determined using fluorescence microscopy. The results indicate that the administration of essential phospholipids leads to a decrease in the cholesterol content in the oocytes' membranes by 13% (p < 0.05). The stiffness of oocytes from flies that received essential phospholipids was 14% higher (p < 0.05) and did not change during 6 h of simulated weightlessness or hypergravity, and neither did the area, which indicates their resistance to deformation. Moreover, the exposure to simulated weightlessness and hypergravity of oocytes from flies that received a standard nutrient medium led to a more intense loss of cholesterol from cell membranes after 30 min by 13% and 18% (p < 0.05), respectively, compared to the control, but essential phospholipids prevented this effect.

High light and nitrogen deficiency synergistically lead to biofuel and biomass accumulation in diatoms

As a clean energy source, the lipids from microalgae serve as alternatives to fossil oils. There are significant differences in the biofuel and biomass accumulation of microalgae under different environmental conditions. This study investigated the response of biofuel accumulation, photosynthetic capacity, and growth of diatom Thalassiosira pseudonana under stress conditions of high light and nitrogen deficiency, aiming to explore the features of biofuel and biomass accumulation in microalgae under special environmental conditions. The results indicated that the synthesis of chlorophyll was inhibited, leading to reduced light absorption and less damage to the photosynthetic system, with an improvement in electron transport efficiency. Lipid metabolism was more active during the dark cycle, with a 2.89-fold accumulation of fatty acids and a significant increase in the content of neutral lipids (2.39 times). The cell cycle progressed faster, resulting in a marked increase in biomass (1.41 times). These processes consumed a considerable amount of energy, affecting the synthesis of ribosomes. These findings suggest that increasing light exposure and reducing nitrogen supply can achieve the co-accumulation of biofuel and biomass in T. pseudonana.

Lipids Mediate Arbuscule Development and Senescence in Tomato Roots Colonized by Arbuscular Mycorrhizae Fungus under Drought Stress.

Arbuscular mycorrhizae (AM) symbiosis can enhance plant resistance to drought stress (DS). This study aimed to investigate the DS effects on lipids at different stages of symbiosis and to link lipid profiles to arbuscule dynamics in tomato roots colonized by AM fungi. DS increased mycorrhizal colonization and arbuscule abundance at an early stage but decreased them at a later stage, delayed arbuscule development, and accelerated arbuscule senescence at a later stage. DS decreased the contents of phospholipids (PLs) and saturated neutral lipids (NLs) at the early stage but increased the contents of saturated PLs and unsaturated NLs at the late stage. Specifically, DS inhibited AM-specific PL contents but increased AM-specific NL contents, which was supported by the expression of RAM2, STR/STR2. These data indicate the negative effect of DS on AM symbiosis and arbuscule dynamics with the effect size depending on the symbiosis stage, which highlights the importance of the symbiosis stage under abiotic stress.

Arbuscular Mycorrhizal Fungi Mediate the Acclimation of Rice to Submergence.

Flooding is a critical factor that limits the establishment of a symbiosis between rice and arbuscular mycorrhizal fungi (AMF) in wetland ecosystems. The distribution of carbon resources in roots and the acclimation strategies of rice to flooding stress in the presence of AMF are poorly understood. We conducted a root box experiment, employing nylon sheets or nylon meshes to create separate fungal chambers that either prevented or allowed the roots and any molecules to pass through. We found that the mycorrhizal colonization rate and the expression of genes OsD14L and OsCERK1, which are involved in fungal perception during symbiosis, both increased in mycorrhizal rice roots following intermittent flooding compared to continuous flooding. Furthermore, AMF inoculation affected root morphological traits, facilitating both shallower and deeper soil exploration. Increased submergence intensity led to carbohydrate deprivation in roots, while high mycorrhizal colonization increased soil oxygen consumption and decreased the neutral lipid concentration in roots. However, mycorrhizal inoculation increased the rice photosynthesis rate and facilitated acclimation to submergence by mediating the expression of the genes OsCIPK15 and OsSUB1A to enhance rice shoot elongation and the sugar concentration in roots as a result of reduced competition for carbon between rice and AMF under different flooding conditions.

Antifungal activity of β-lapachone against a fluconazole-resistant Candida auris strain.

Candida spp. can be found in the human microbiome. However, immunocompromised patients are likely to develop invasive Candida infections, with mortality rates higher than 50%. The discovery of C. auris, a species that rapidly acquire antifungal resistance, increased the concern about Candida infections. The limited number of antifungal agents and the high incidence of resistance to them make imperative the development of new antifungal drugs. β-lapachone is a biological active naphthoquinone that displays antifungal activity against C. albicans and C. glabrata. The aim of this study was to evaluate if this substance affects C. auris growth and elucidate its mechanism of action. A fluconazole-resistant C. auris isolate was used in this study. The antifungal activity of β-lapachone was determined through microbroth dilution assays, and its mechanism of action was evaluated using fluorescent probes. Interaction with fluconazole and amphotericin B was assessed by disk diffusion assay and checkerboard. β-lapachone inhibited planktonic C. auris cell growth by 92.7%, biofilm formation by 84.9%, and decrease the metabolism of preformed biofilms by 87.1% at 100µg/ml. At 100µg/ml, reductions of 30% and 59% of Calcofluor White and Nile red fluorescences were observed, indicating that β-lapachone affects cell wall chitin and neutral lipids content, respectively. Also, the ratio 590nm/529nm of JC-1 decreased 52%, showing that the compound affects mitochondria. No synergism was observed between β-lapachone and fluconazole or amphotericin B. Data show that β-lapachone may be a promising candidate to be used as monotherapy to treat C. auris resistant infections.

Maternal Exercise Improves Infant Mesenchymal Stem Cell Lipid Storage and Oxidation, and Decreases Infant Adiposity

Rates of pediatric obesity are continuously rising and are likely to translate into high incidence of metabolic disease later in life. Maternal exercise during pregnancy has been established as a useful non-pharmacological intervention to improve infant metabolic health; however, mechanistic insight behind these adaptations remains mostly confined to animal models. Infant mesenchymal stem cells (MSCs) give rise to a multitude of infant peripheral tissue (e.g., skeletal muscle) and remain involved in mature tissue repair and maintenance. Importantly, these cells preserve the phenotype of an offspring donor and represent a model for the investigation of mechanisms behind infant metabolic health improvements. We used undifferentiated MSC collected during a randomized controlled trial to investigate if maternal exercise, regardless of exercise mode, affects infant adiposity and fatty acid oxidation. Compared to control, we observed &gt;160% higher neutral lipid content in Ex-MSCs (p&lt;0.01). However, this finding was coupled with significantly higher complete fatty acid oxidation to CO2 and lower incomplete fat oxidation (ASM production) in Ex-MSCs (p≤0.05). Infants in the exercise group had significantly lower body fat percentage compared to same age infants of controls at one month of age (p≤0.05). There was no difference in lean body mass volume (p&gt;0.05) or infant one-month BMI (p=0.069). Together, these data indicate that maternal exercise exposure in utero is still associated with decreased infant adiposity one month after birth. MSC lipid content was positively associated with maternal fitness and exercise volume (p&lt;0.01) during pregnancy. Finally, an increase in average maternal exercise volume (MET*min/wk; r=-0.4, p≤0.05) and total gestational MET*min (r=-0.36, p≤0.05), but not VO2max (p=0.07) was associated with infant 1-month body fat percentage. Together, these data showcase the influence of maternal exercise exposure on improvements in cellular metabolism and at 1 month of age. Collectively, our data suggests that exposure to maternal exercise could contribute to lower adiposity in early infancy. This project was supported by the American Heart Association (18IPA34150006 to L.E.M.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Waterborne exposure to the antineoplastic 5-fluorouracil alters lipid composition in larval zebrafish (Danio rerio)

Antineoplastic medications are present in aquatic environments and are measured at relatively high concentrations in hospital sewage effluent. Thus, it is important to characterize risk associated with waterborne exposures to anticancer drugs. The drug 5-fluorouracil (5-FU) is used to treat several types of cancers, acting to inhibit cell division and cellular metabolism. The objectives of this study were to determine the effects of 5-FU on developmental endpoints and lipid composition in zebrafish. 5-FU did not negatively affect development nor survival in developing zebrafish at concentrations up to 1000 μg/L. However, 5-FU increased neutral lipid content in zebrafish larvae, indicating potential for lipid dysregulation. To further discern effects on lipids, lipidomics was conducted and a total of 164 lipids belonging to 14 lipid classes were identified. Significant changes (false discovery rate < 0.05) in abundance were detected for 19 lipids including some ceramides, ether-linked phosphatidylethanolamines, and sphingomyelins among others. We also measured the expression levels of 14 lipid-related enzymes and transporters (e.g., acox3, dgat1, fads2, fasn, elovl2) using real-time PCR; however, mRNA abundance levels were not affected, suggesting transcriptional changes may not be a primary mechanism underlying lipid dysregulation. Locomotor activity was measured in zebrafish as lipids are needed for swimming activity in larvae. Exposure to 5-FU did not affect locomotor activity up to 1000 μg/L. We conclude that lipids accumulate in larval zebrafish with exposure to 5-FU, which can subsequently affect lipid composition. These data reveal potential lipid signatures of 5-FU exposure and contribute to risk assessments for antineoplastic exposure in aquatic environments.

Characterizing interregional differences in the rheological properties and composition of rat small intestinal mucus.

The mucus layer in the small intestine is generally regarded as a barrier to drug absorption. However, the mucus layer is a complex system, and presently, only a few studies have been conducted to elucidate its physicochemical properties. The current study hypothesizes that the mucus layer contains solubility-enhancing surfactants and thus might aid the oral absorption of poorly water-soluble drugs.Mucus was sampled from sections of the small intestine of fasted rats to analyze the rheological properties and determine the mucus pH and concentrations of proteins and endogenous surfactants, i.e., bile salts, polar lipids, and neutral lipids.The mucus layer in the two proximal sections of the small intestine exhibited different rheological properties such as higher zero-shear viscosity and lower loss tangent and higher protein concentrations compared to all subsequent sections of the small intestine. The pH of the mucus layer was stable at ~ 6.5 throughout most of the small intestine, but increased to 7.5 in the ileum.The bile salt concentrations increased from the duodenum (16.0 ± 2.2mM) until the mid jejunum (55.1 ± 9.5mM), whereas the concentrations of polar lipids and neutral lipids decreased from the duodenum (17.4 ± 2.2mM and 37.8 ± 1.6mM, respectively) until the ileum (4.8 ± 0.4mM and 10.7 ± 1.1mM, respectively).In conclusion, the mucus layer of the rat small intestine contains endogenous surfactants at levels that might benefit solubilization and absorption of orally administered poorly water-soluble drugs.

We Must Abandon the Myth: Oxidized Low-density Lipoprotein is not a Lipoprotein that Plays a Key Role in Atherogenesis.

The purpose of this review is to revisit in detail the arguments supporting or disproving the hypothesis that oxidized low-density lipoprotein (LDL) plays a key role in atherosclerotic lesion development. The detection of oxidized LDL in vivo was extremely important for confirming its key role in atherogenesis. Indirect evidence of its existence included the presence of autoantibodies against malondialdehyde-treated LDL in human blood; however, the affinity of circulating antibodies to another LDL modification, such as desialylated LDL, was an order of magnitude stronger. At least 3 forms of atherogenic modified lipoproteins were isolated from the blood of atherosclerotic patients using different methods, namely, small dense, electronegative and desialylated. Their properties were so similar that it was suggested that the three types could be classified as the same multiple-modified LDL particle. It has been shown that when native (unmodified) LDL is incubated with autologous serum from patients with atherosclerosis, multiple modifications occur, which include desialylation, a decrease in the content of phospholipids and neutral lipids, a decrease in particle size, an increase in negative charge and other physical and chemical changes. Longer incubation also increased the susceptibility of LDL to oxidation. Thus, LDL oxidation is not the only, much less the most important, form of atherogenic modification of LDL since it occurs at the last stages of multiple modifications cascade and does not significantly increase the atherogenic potential of multiple-modified LDL. Finally, clinical trials did not support the oxidative hypothesis; however, research on oxidized LDL continues, influencing the future research. It is time to abandon the myth!

Lipid spectrum of cell membranes and blood plasma in adolescents with recurrent cephalgia associated with arterial hypertension. Methods of correction

Purpose. To study the characteristics of the content of neutral lipids and phospholipid fractions in the membranes of erythrocytes and blood plasma of adolescents with cephalalgia and arterial hypertension (AH). Materials and methods. Subject of the study – adolescents 12–17 years old (boys and girls) with cephalgia and the presence of AH (main group) and without AH (comparison group). Methods: questionnaires, laboratory, statistical. Examination program: verification of the clinical form of cephalgia (episodic, chronic); assessment of the presence of AH (using percentile tables, taking into account age, gender and height percentile); determination of the concentration of neutral lipids and phospholipid fractions in blood plasma and erythrocyte membranes using thin layer chromatography. Results. Of the 64 examined adolescents – patients of the pediatric department with recurrent cephalgia, the frequent (chronic) form of cephalgia was verified in 40 (62.5%) people, AH was diagnosed in 10 (25.0%) adolescents with chronic cephalgia and in 30 (75.0%) adolescents with this form of cephalalgia AH were not identified. Features of the lipid spectrum of adolescents with a chronic form of cephalgia associated with AH included a lower concentration of total lipids and triglycerides in erythrocyte membranes, higher values of the general phospholipids/free cholesterol ratio and a smaller number of statistically significant correlations. Conclusion. The features we identified in the lipid spectrum of erythrocyte membranes and blood plasma in adolescents with a frequent (chronic) form of cephalalgia associated with AH may indicate the presence in adolescents of initial minimal changes in the biochemical status of cell membranes at the stages of development of cardiovascular pathology – hypertension and coronary heart disease.

Assessment of Full-Fat Tenebrio molitor as Feed Ingredient for Solea senegalensis: Effects on Growth Performance and Lipid Profile.

Tenebrio molitor (TM) is considered as one of the most promising protein sources for replacing fish meal in aquafeeds, among other things because it is rich in protein, a good source of micronutrients and has a low carbon footprint and land use. However, the main drawback of TM is its fatty acid profile, in particular its low content of n-3 PUFA. This study evaluates the effects of partially replacing plant or marine-derived with full-fat TM meal at two different levels on growth performance and lipid profiles of Senegalese sole (Solea senegalensis). For this purpose, a control diet (CTRL) and four experimental isoproteic (53%) and isolipidic (16%) diets were formulated containing 5 and 10% TM meal replacing mostly fish meal (FM5 and FM10), or 10 and 15% TM meal replacing mostly plant meal (PP10 and PP15). Fish (215 g) were fed at 1% of their body weight for 98 days. The final body weight of fish fed the experimental diets containing TM meal was not different from that of fish fed the CTRL diet (289 g). However, the inclusion of TM meal resulted in a gradual improvement in growth rate and feed efficiency in both cases (replacement of fish or plant meals), and significant differences in specific growth rate (SGR) were observed between fish fed the CTRL diet (SGR = 0.30% day-1) and those fed diets with the highest TM meal content (PP15; SGR = 0.35% day-1). The experimental groups did not show any differences in the protein content of the muscle (19.6% w/w). However, significant differences were observed in the total lipid content of the muscle, with the FM10, PP10, and PP15 groups having the lowest muscle lipid contents (2.2% ww). These fish also showed the lowest neutral lipid content in muscle (6.6% dw), but no differences were observed in the total phospholipid content (2.6% dw). Regarding the fatty acid profile, fish fed FM10, PP10 and PP15 had lower levels of linoleic acid (18:2n-6) and higher levels of oleic acid (18:1n-9) in liver and muscle compared to fish fed CTRL. However, no differences were found between fish fed CTRL and TM-based diets for docosahexaenoic acid (22:6n-3) and total n-3 PUFA in liver and muscle. In conclusion, our study demonstrated that full-fat TM inclusion up to 15% in S. senegalensis diets had no negative effects or even some positive effects on fish survival, growth performance, nutrient utilization and flesh quality.

Clinical and functional features and quality of life in various phenotypes of occupational asthma

Purpose. To study the characteristics of the content of neutral lipids and phospholipid fractions in the membranes of erythrocytes and blood plasma of adolescents with cephalalgia and arterial hypertension (AH). Materials and methods. Subject of the study – adolescents 12–17 years old (boys and girls) with cephalgia and the presence of AH (main group) and without AH (comparison group). Methods: questionnaires, laboratory, statistical. Examination program: verification of the clinical form of cephalgia (episodic, chronic); assessment of the presence of AH (using percentile tables, taking into account age, gender and height percentile); determination of the concentration of neutral lipids and phospholipid fractions in blood plasma and erythrocyte membranes using thin layer chromatography. Results. Of the 64 examined adolescents – patients of the pediatric department with recurrent cephalgia, the frequent (chronic) form of cephalgia was verified in 40 (62.5%) people, AH was diagnosed in 10 (25.0%) adolescents with chronic cephalgia and in 30 (75.0%) adolescents with this form of cephalalgia AH were not identified. Features of the lipid spectrum of adolescents with a chronic form of cephalgia associated with AH included a lower concentration of total lipids and triglycerides in erythrocyte membranes, higher values of the general phospholipids/free cholesterol ratio and a smaller number of statistically significant correlations. Conclusion. The features we identified in the lipid spectrum of erythrocyte membranes and blood plasma in adolescents with a frequent (chronic) form of cephalalgia associated with AH may indicate the presence in adolescents of initial minimal changes in the biochemical status of cell membranes at the stages of development of cardiovascular pathology – hypertension and coronary heart disease.

Lipid spectrum of cell membranes and blood plasma in adolescents with recurrent cephalgia associated with arterial hypertension. Methods of correction

Purpose. To study the characteristics of the content of neutral lipids and phospholipid fractions in the membranes of erythrocytes and blood plasma of adolescents with cephalalgia and arterial hypertension (AH). Materials and methods. Subject of the study – adolescents 12–17 years old (boys and girls) with cephalgia and the presence of AH (main group) and without AH (comparison group). Methods: questionnaires, laboratory, statistical. Examination program: verification of the clinical form of cephalgia (episodic, chronic); assessment of the presence of AH (using percentile tables, taking into account age, gender and height percentile); determination of the concentration of neutral lipids and phospholipid fractions in blood plasma and erythrocyte membranes using thin layer chromatography. Results. Of the 64 examined adolescents – patients of the pediatric department with recurrent cephalgia, the frequent (chronic) form of cephalgia was verified in 40 (62.5%) people, AH was diagnosed in 10 (25.0%) adolescents with chronic cephalgia and in 30 (75.0%) adolescents with this form of cephalalgia AH were not identified. Features of the lipid spectrum of adolescents with a chronic form of cephalgia associated with AH included a lower concentration of total lipids and triglycerides in erythrocyte membranes, higher values of the general phospholipids/free cholesterol ratio and a smaller number of statistically significant correlations. Conclusion. The features we identified in the lipid spectrum of erythrocyte membranes and blood plasma in adolescents with a frequent (chronic) form of cephalalgia associated with AH may indicate the presence in adolescents of initial minimal changes in the biochemical status of cell membranes at the stages of development of cardiovascular pathology – hypertension and coronary heart disease.

Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

Evaluation of the T-Cell Metabolic State of Starting Material and Manufactured CAR T Cell Products and Clinical Outcome of CAR T-Cell Therapy for Large B Cell Lymphoma

Background Although chimeric antigen receptor (CAR)-T cells are a viable treatment option for patients with relapsed or refractory (r/r) aggressive B-cell lymphomas, more than half of patients experience relapse within the first two years after product infusion. Successful autologous cell-based anti-cancer therapies require persistence of effector cells, features that highly depend on complex metabolic processes. We earlier demonstrated in chronic lymphocytic leukemia, that expansion and persistence of CD19-CAR T-cell infusion in vivo was correlated with increased mitochondrial mass and enhanced biogenesis(van Bruggen et al, Blood 2019). Metabolic features might therefore serve as early biomarker tools to predict efficacy and outcome of CAR T-cell therapy and might allow therapeutic targeting but did was never studied in a prospective clinical trial. Aims Perform extensive phenotyping and state-of-the-art metabolic state analyses of T cells prior and post CAR T cell production and investigate correlations with clinical outcomes and patient characteristics. Methods Samples were obtained from large B cell lymphoma (LBCL) patients that were part of the TRANSCEND-NHL-001 trial (NCT02631044, Abramson et al, Lancet 2020).Thirty-two patients with r/r B-cell lymphoma who received CAR-T cell treatment lisocabtagene maraleucel (16 responders (R) and 16 non-responders (NR)) were included in our analyses and (metabolically) analyzed by flow cytometry and extracellular flux analysis. Metabolic phenotyping was performed on 53 samples at baseline and 44 samples after an in vitro stimulation. Extracellular flux analysis was done on 14 DP samples. The cohort was split evenly between Responders and Non-responders, and balanced for a score calculated taking key clinical baseline characteristics that were found to be associated with response into account (NCT02631044, Abramson et al, Lancet 2020). Both T cells obtained at time of leukopheresis (starting material (SMAT)) and CAR-T drug products (DP), (following transduction of a CD19-targeted CAR T cell product with a defined CD4 and CD8 composition bearing a 4-1BB costimulatory domain and expansion) were analyzed directly after thawing and after stimulation with anti-CAR-antibody in case of CDP, or αCD3/αCD28 antibodies for SMAT samples. Patients consented to these studies and researchers were blinded for clinical outcomes at time of analysis. Results We found that the baseline clinical factors that associate with clinical response are related to specific metabolic cellular phenotypes, both in SMAT and DP, and with and without T cell stimulation. SMAT samples from patients with higher baseline scores show signs of an increased exhausted phenotype with regards to cellular ROS and exhaustion marker expression, whilst a more plentiful memory-like phenotype was seen in patients with a lower baseline score. When clustering by clinical response status, analysis of glucose and fatty-acid uptake, neutral lipid content, cellular ROS, mitochondrial mass and potential and flux data showed that responders, in both SMAT and DP samples, have an all-round increased metabolic activity upon αCD3/αCD28 or CAR T-receptor triggering, respectively, when compared to non-responders (Figure 1A). Furthermore, responding patients show enrichment of CD27 expression in the SMAT material, indicating a more abundant memory phenotype. Summary/Conclusions This is the first evidence of an association between a specific immune-metabolic phenotype of (CAR-) T cells and outcome of CAR-T cell therapy in patients with relapsed/refractory LBCL. More specifically, a higher abundance of memory phenotypes, metabolic activity and mitochondrial biogenesis of both harvested T cells and CAR-T product associates with improved clinical response. Combining metabolic, phenotypical and clinical characteristics and biomarkers of patient T cells (both pre- or post-production) shows the importance of the metabolic phenotype and fitness of T cell starting material, underlying the importance of early line CAR T therapy following diagnosis.

Naphthalene acetic acid and scorbic acid significantly improve neutral lipid accumulation in Chlorella species under nutrient limitations

Nutrient starvation condition has already been exploited as a suitable strategy to induce more lipid production. In this study, nutrient limitation has been coupled with phytohormone or antioxidant supplementation to identify the potential of chemical stimulants to support growth and neutral lipid accumulation. Naphthalene acetic acid (NAA), and Ascorbic acid (AA), at concentrations of 5, 10, and 15 ppm were supplemented to N-limited level 1 (0.25 g NO3-N/L), N-limited level 2 (0.041 gNO3-N/L), and P-limited (0.01 gPO4-P/L) culture media of two Chlorella species to maximize biomass and lipid production. The results show that neutral lipid content increases 70 % when AA is added to the P-limited medium of Chlorella vulgaris IG-R-96 at 15 ppm. At N-limitation level 1 coupled with NAA and AA supplementations at 15 ppm, neutral lipid contents increase 107 % and 128 % in C. vulgaris IG-R-96, and 42 % and 60 % in Chlorella sorokiniana IG-W-96, respectively. When simultaneous N- and P-limitation is applied, neutral lipid content becomes almost double in C. vulgaris IG-R-96 at 10 ppm of NAA supplement. The effects of phytohormone and antioxidant on growth and neutral lipid accumulation is species-dependent and could be more effective when it is combined with nutrient limitation conditions. Stimulant supplementation is significantly effective for neutral lipid accumulation.

Seasonal lipid dynamics of four Arctic bivalves: Implications for their physiological capacities to cope with future changes in coastal ecosystems.

The Arctic is exposed to unprecedented warming, at least three times higher than the global average, which induces significant melting of the cryosphere. Freshwater inputs from melting glaciers will subsequently affect coastal primary production and organic matter quality. However, due to a lack of basic knowledge on the physiology of Arctic organisms, it remains difficult to understand how these future trophic changes will threaten the long-term survival of benthic species in coastal habitats. This study aimed to gain new insights into the seasonal lipid dynamics of four dominant benthic bivalves (Astarte moerchi, Hiatella arctica, Musculus discors, and Mya truncata) collected before and after sea ice break-up in a high-Arctic fjord (Young Sound, NE Greenland). Total lipid content and fatty acid composition of digestive gland neutral lipids were analyzed to assess bivalve energy reserves while the fatty acid composition of gill polar lipids was determined as a biochemical indicator of interspecies variations in metabolic activity and temperature acclimation. Results showed a decrease in lipid reserves between May and August, suggesting that bivalves have only limited access to fresh organic matter until sea ice break-up. The lack of seasonal variation in the fatty acid composition of neutral lipids, especially essential ω3 fatty acids, indicates that no fatty acid transfer from the digestive glands to the gonads occurs between May and August, and therefore, no reproductive investment takes place during this period. Large interspecies differences in gill fatty acid composition were observed, which appear to be related to differences in species life span and metabolic strategies. Such differences in gill fatty acid composition of polar lipids, which generally influence metabolic rates and energy needs, may imply that not all benthic species will be equally sensitive to future changes in primary production and organic matter quality in Arctic coastal habitats.

Metabolic interpretation of NaCl stress-induced lipid accumulation in microalgae for promising biodiesel production with saline wastewater

The rising use of saline wastewater for biodiesel production through oleaginous microalgae demands fundamental studies on the metabolic response of microalgal cells to salt stress. Thus, we investigated how salt stress promotes lipid accumulation inside Botryococcus sp. NJD-1 by tracking critical metabolites and proteases as well as analyzing the cellular redox balance. The results showed that the lipid content of microalgal cells followed a negative binomial correlation with intracellular reactive oxygen species (ROS), while the neutral lipid content increased linearly with salinity in range of 0–20 g/L NaCl. The optimal lipid production was achieved as 54.5 % and 110.5 mg/L d-1 at 10 g/L NaCl. Further comparing the metabolomics and proteomics data at 0 and 10 g/L NaCl, the adenosine triphosphate (ATP) and nicotinamide adenine nucleotide phosphate (NADPH) were found significantly increased to quench the Na+-induced ROS. The created NADPH and ATP accord the enhanced activity of the upper Pyruvate-Malate cycle and tricarboxylic acid cycle (TCA) cycle. Therefore, ROS detoxification is proposed to be the essential mechanism for microalgal lipid accumulation under salt stress. The cellular triacylglycerol (TAG) synthesis triggered by salinity is mainly mediated by an acetyl-CoA-dependent Kennedy pathway. These findings will deepen our understanding of the nexus of ambient salt, intracellular ROS and lipid metabolism for microalgae and advance the exploit of saline wastewater for microalgal lipid production.