Higher Intracellular Lipid Content Research Articles

Lipid profiling in chilled coral larvae

Coral reefs are disappearing worldwide as a result of several harmful human activities. The establishment of cryobanks can secure a future for these ecosystems. To design effective cryopreservation protocols, basic proprieties such as chilling tolerance and lipid content must be assessed. In the present study, we investigated chilling sensitivity and the effect of chilling exposure on the lipid content and composition of larvae belonging to 2 common Indo-Pacific corals: Seriatopora caliendrum and Pocillopora verrucosa. The viability of coral larvae incubated with 0.5, 1, and 2 M ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (Me2SO), methanol, or glycerol and kept at 5 °C for different time periods was documented. In addition, we investigated the content of cholesterol, triacylglycerol (TAG), wax ester (WE), sterol ester (SE), lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and several fatty acid (FA) classes in coral propagules incubated with 1 M PG or EG and kept at 5 °C for 6 h. Moreover, we examined seasonal changes in the aforementioned lipid classes in coral larvae. S. caliendrum incubated with 0.5 M PG or Me2SO and chilled for 2 h exhibited a viability rate of 11 ± 11%, whereas P. verrucosa exhibited a viability rate of 22 ± 14% after being chilled for 4 h. Furthermore, the results indicated that chilling exposure did not affect the content of any investigated lipid class in either species. The higher concentration of SE in P. verrucosa compared to S. caliendrum larvae may have contributed to the different cryotolerance displayed by the 2 larval species. A year-round lipid analysis of both coral larvae species revealed trends of homeoviscous adaptation and seasonal enhancement of lipid fluxes from symbionts to the host. During winter, the cholesterol/phospholipid ratio significantly increased, and P. verrucosa larvae exhibited an averagely decrease in FA chain lengths. During spring and summer, intracellular lipid content in the form of TAGs and WEs significantly increased in both species, and the average content of Symbiodiniaceae-derived FAs increased in P. verrucosa larvae. We concluded that the low cryotolerance displayed by S. caliendrum and P. verrucosa larvae is attributable to their chilling-sensitive membrane lipid profile and the high intracellular lipid content provided by their endosymbionts.

Direct and Label-Free Cell Status Monitoring of Spheroids and Microcarriers Using Microfluidic Impedance Cytometry.

3D cellular spheroids/microcarriers (100µm-1mm) are widely used in biomanufacturing, and non-invasive biosensors are useful to monitor cell quality in bioprocesses. In this work, a novel microfluidic approach for label-free and continuous-flow monitoring of single spheroid/microcarrier (hydrogel and Cytodex) based on electrical impedance spectroscopy using co-planar Field's metal electrodes is reported. Through numerical simulation and experimental validation, two unique impedance signatures (|ZLF | (60kHz), |ZHF | (1MHz)) which are optimal for spheroid growth and viability monitoring are identified. Using a closed-loop recirculation system, it is demonstrated that |ZLF | increases with breast cancer (MCF-7) spheroid biomass, while higher opacity (impedance ratio |ZHF |/|ZLF |) indicates cell death due to compromised cell membrane. Anti-cancer drug (paclitaxel)-treated spheroids also exhibit lower |ZLF | with increased cell dissociation. Interestingly, impedance characterization of adipose-derived mesenchymal stem cell differentiation on Cytodex microcarriers reveals that adipogenic cells (higher intracellular lipid content) exhibit higher impedance than osteogenic cells (more conductive due to calcium ions) for both microcarriers and single cell level. Taken together, the developed platform offers great versatility for multi-parametric analysis of spheroids/microcarriers at high throughput (≈1 particle/s), and can be readily integrated into bioreactors for long-term and remote monitoring of biomass and cell quality.

The Porcine Experimental Model for Oocyte Cryopreservation by Vitrification

Pigs have anatomical-physiological similarities with the human in several aspects, mainly in the reproduction function, which is of great interest in this review. However, in oocytes, there are differences between both species, impacting the vitrification success. For example, porcine oocytes have higher intracellular lipid content than women oocytes, affecting cryoprotectant permeability and increasing ice crystal formation. Despite these differences, the porcine model has been important for the development of assisted reproduction technologies in humans, as well as in the genetic improvement of livestock.

Abstract C109: Tumor derived exosomal lipids-induced dendritic cell immunosuppression by PPARα

Abstract Dendritic cells (DCs), a type of professional antigen-presenting cells, play a key role in orchestrating anti-tumor immune responses. In the tumor microenvironment, tumor infiltrating DCs (TIDCs) present tumor associated antigens to effector T cells and facilitate the induction of memory T cells to prevent tumor recurrence. However, emerging evidences indicate that TIDCs become immunosuppressive with a high intracellular lipid content phenotype. TIDCs with abnormal lipid accumulation are associated with endoplasmic reticulum (ER) stress responses or increased fatty acid oxidation which also contribute to immunosuppressive. However, the critical factor inducing lipid-laden immunosuppressive TIDCs remains to be determined. Here we report: (a) In vitro, tumor derived exosomes (TDEs), isolated from tumor culture medium can directly induce lipid accumulation in DCs rather than TDE-free tumor culture medium. In vivo, we generate GFP-CD9-tumor cells to trace the receiver cells of GFP-labeled TDEs in the tumor microenvironment. The results show that TIDCs capture GFP-labeled TDEs and become lipid abundant. By treating DCs with TDEs from different tumor cell lines and of different concentrations or time, we observe that TDE-induced lipid accumulation in DCs is tumor-origin independent but time and concentration dependent. Collectively, we can conclude that TDEs are responsible for the intracellular lipid accumulation in DCs. (b) Upon internalization of TDEs, increased expression of genes involved in lipid accumulation and higher mitochondrial respiration are observed in DCs, which results in lower ability to prime tumor-specific T cell responses. (c) Then, we demonstrate that the fatty acids contained in TDEs directly contribute to lipid laden DC, rather than proteins or nucleic acids. Moreover, we identify PPARα as the key player in orchestrating both lipid metabolism and immunological features of DCs. (d) PPARα initiates both de novo lipid synthesis and fatty acids β-oxidation in DCs by sensing the excess fatty acids, which leads to immunosuppression in DCs. Pharmacological inhibitor of PPARα can overcome TDE-induced lipid accumulation, fatty acids β-oxidation and immunosuppressive function in DCs. (e) Combination therapy of PPARα pharmacological inhibitor and immunotherapy (such as the check point blocker or the therapeutic cancer vaccine) shows superior anti-tumor efficacy over monotherapy. The present study unprecedentedly provides substantial evidences that TDEs can act as a major immunosuppressive component within tumor microenvironment to undermine the proper functions of TIDCs. More importantly, we demonstrate targeting PPARα could be a potential strategy to design novel immunotherapy combinations, in order to maximize the induction, expansion and cytotoxicity of tumor-specific CD8+ T cells by restoring TIDCs function. Citation Format: Xiaozhe Yin, Wenfeng Zeng, Bowen Wu, Zihao Wang, Hongjian Tian, Luyao Wang, Luoyang Wang, Xiuli Wei, Yan Qin, Fayun Zhang, Chunling Zhang, Lingtao Jin, Wei Liang. Tumor derived exosomal lipids-induced dendritic cell immunosuppression by PPARα [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C109. doi:10.1158/1535-7163.TARG-19-C109

Simultaneous Production of Lipids and Carotenoids by the Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater

The objective of this study was to determine the possibility of simultaneous biosynthesis of lipids and carotenoids by the Rhodotorula yeast strains in media with waste glycerol and deproteinized potato wastewater and to determine the level of pollution reduction by media. On the basis of results obtained during the yeast microcultures in the Bioscreen C system, it was found that potato wastewater and glycerol can be used as components of media for Rhodotorula glutinis, Rhodotorula mucilaginosa, and Rhodotorula gracilis yeast strains. The amount of glycerol added to media higher than 10% significantly decreased the growth rate of yeast. The results of yeast culture in the laboratory shaker flasks showed a possibility of simultaneous production of lipids and carotenoids by R. glutinis, R. mucilaginosa, and R. gracilis yeast strains during cultivation in media containing only waste glycerol and deproteinized potato wastewater. A higher intracellular lipid content (approximately 15 g/100 gd.w.) was obtained for R. mucilaginosa and R. gracilis yeast biomass after cultivation in experimental media with waste glycerol and potato wastewater. In conclusion, the yeast grown in media with potato wastewater supplemented with 3% or 5% glycerol synthesized carotenoids, and their content in biomass did not exceed 230 μg/gd.w.

Exploration of sodium lignosulphonate's effects on lipid production by Rhodosporidium toruloides

Microbial oil is drawing increasing interest all over the world as an alternative non-food feedstock for biodiesel production. Rhodosporidium toruloides has been considered as a promising candidate to convert glycerol into microbial oils due to its good substrate adaptability, high intracellular lipid content, and the potential of co-production of some pigments. Interestingly, we have also found that sodium lignosulphonate (SL) could significantly enhance the cell growth and lipid accumulation of R. toruloides. In the present work, we investigated the possible mechanisms for the enhancement of lipid production by SL. It was found that SL could not be utilized by R. toruloides as a carbon source. No apparent changes on the morphology of cell membranes and the fatty acid profiles of microbial lipids were found when SL was added. Further experiments indicated that the volumetric oxygen transfer coefficient was increased by nearly 15% on average by adding 4g/L SL, which might be the main mechanism for the improvement of lipid production.

Birth of a domestic cat kitten produced by vitrification of lipid polarized in vitro matured oocytes

The ability to cryopreserve oocytes is an effective method to retain valuable genetic material of mammals, including that of endangered animals. Embryos of domestic cats are amenable to cryopreservation, whereas their oocytes are much less cryo-tolerant. The capability of oocytes to survive cryopreservation is affected by several factors, one of which has been hypothesized to be the high concentration of intracellular lipids. To test this hypothesis, in this study we polarized lipids of cat oocytes and tested their cooling and freezing sensitivity. We found that the sensitivity of oocytes to cooling and cryopreservation does appear to be related to their high intracellular lipid content, as indicated by higher cryosurvival and development into blastocysts when intracellular lipids of in vitro matured oocytes were polarized before vitrification. However, polarization of all intracellular lipids was detrimental to development of embryos. Cell numbers in blastocysts derived from fully polarized/vitrified oocytes were significantly lower than those of partially polarized/vitrified or non-vitrified/fresh oocytes. Although embryos derived from fully polarized/vitrified oocytes developed to the blastocyst stage at higher rates than those of partially polarized/vitrified or non-centrifuged/vitrified oocytes, their in vivo developmental competence was compromised. When embryos derived from fully polarized/vitrified oocytes were transferred, although two recipients became pregnant, all implanted embryos were reabsorbed. In contrast, when embryos derived from oocytes that were only partially lipid polarized before vitrification and then were transferred, one recipient did become pregnant and produced a live healthy kitten. The present results suggest that other approaches to altering intra-cellular lipid levels in cat oocytes should be evaluated to improve their functional survival after cryopreservation.

Abstract 87: Sterol-Mediated Regulation of γδ T Lymphocytes

Objective: gammadelta (γδ) T lymphocytes, which bridge innate and adaptive immunity, may be differentially regulated by cholesterol and play distinct roles from the conventional alphabeta (αβ) subset in the development of atherosclerosis. γδ T cells represent a small subset of total T cells (∼ 3-5% of CD3+ cells). These cells express unique γδ T cell receptors, and recognize lipid and peptide antigens without processing and presentation by MHC molecules. γδ T cells react much faster toward pathogen invasion than αβ T cells, and thus are considered one of the first lines of immune defense. Methods: αβ and γδ T lymphocytes from mice were evaluated for their lipid content, gene expression, proliferation, apoptosis, and activation. Results and conclusion: Nile Red staining revealed higher intracellular lipid content in γδ T cells, comparing to the αβ subset (1.3 folds of αβ, p<0.05; n=5). We therefore hypothesize that the basal higher sterol content elicits the turnover and activation of γδ T lymphocytes for rapid response. We found that genes involved in cholesterol esterification (ACAT1 and ACAT2) and efflux (ABCA1) are upregulated in γδ cells compared to conventional αβ T cells. PCSK9, which downregulates LDLR posttranslationally, is elevated in γδ T cells, suggesting cholesterol metabolism is differentially regulated in the two subsets. BrdU incorporation and annexin V staining revealed significantly higher proliferative (3.4 folds of αβ, p<0.001; n=8) and apoptotic (34.9 folds of αβ, p<0.001; n=6) rates of γδ T cells in vivo, suggesting a higher turnover rate of the γδ subset. Staining for ganglioside GM1 showed elevated lipid raft levels in γδ T cells (1.9 folds of αβ, p<0.001; n=8), implying enhanced TCR signaling and activation. Notably, the percentages of total (2.3 folds of αβ, p<0.01; n=3) and pro-inflammatory cytokine IL-17- producing (3.9 folds of αβ, p<0.01; n=4-5) γδ T cells increase in ApoE -/- mice fed a high cholesterol, high fat diet, implying a high sterol/high fat environment may promote γδ T cells activation in vivo. Taken together, our data suggest γδ T cells are more sensitive to cholesterol, and a high sterol environment may enhance γδ T cell turnover and activation in vivo.

106 USING A BUOYANT DENSITY GRADIENT AND NILE RED STAINING TO EVALUATE THE LIPID CONTENT OF BOS TAURUS AND BOS INDICUS OOCYTES

Bos indicus embryos have a lower survival rate compared with Bos taurus after cryopreservation. It has been hypothesized that the lower survival rate is due to higher intracellular lipid content. The objective of this study was to determine if there is a difference in intracellular lipid content of oocytes from mature purebred Brahman and Angus cows. Donor females used in the study were maintained on pasture prior to the onset of the experiment and on a grain-supplemented hay ration during the study. Oocytes were collected from cows at 30-day intervals (3 aspirations/donor) by transvaginal ultrasound-guided oocyte aspirations (TUGA). Mature oocytes were evaluated using a sucrose step gradient procedure and Nile Red staining. FSH (Folltropin-V®; Bioniche Animal Health, Beltsville, Ontario, Canada) administration began on Day 4 of the estrous cycle (estrus = Day 0) twice daily for 3 days in decreasing doses (Brahman 232 mg and Angus 280 mg total), and on Day 8 oocytes were recovered. The mean number of follicles aspirated/donor and oocytes recovered/donor were 20 and 16.61 oocytes for the Brahman donors (n = 6) and 12 follicles and 7.06 oocytes/donor for the Angus donors (n = 10). Oocytes (individual donor basis) were then incubated in TCM-199 supplemented with 10% fetal bovine serum + bLH and bFSH (0.01 U mL−1) at 38.5°C. After 20 h, mature oocytes were denuded by vortexing for 3 min in HEPS + BSA (4 mg mL−1). Buoyancy was tested for individual mature oocytes using a sucrose step density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% sucrose (indicating high lipids) to 35% sucrose (indicating lower lipids). Oocytes recovered from the sucrose were fixed for 24 h in paraformaldehyde for evaluation with Nile Red stain. Oocytes were stained for 24 h, and then placed in Prolong® Gold (Invitrogen, Carlsbad, CA, USA) and evaluated under fluorescence. Oocytes images were evaluated using a Scion Image camera (Scion Corp., Frederick, MD, USA) to calculate mean (± SE) Nile Red units (NRU) (higher NRU = higher lipid content). Treatment groups were analyzed using one-way ANOVA. In summary, Brahman M-II oocytes had significantly lower (P ≤ 0.05) buoyant density, with a significantly higher mean NRU score, when compared with oocytes harvested from Angus donors (Table 1). Based on these results, Brahman oocytes have a higher intracellular lipid content then Angus oocytes. Table 1.Percent sucrose levels and Nile Red units for bovine oocytes from three replicates per donor

Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells

In order to get the high liquefaction yield from marine algae cell mass to fuel oil, the effect of salt stress on the accumulation of lipids and triacylglyceride in Dunaliella cells was investigated. Although initial NaCl concentration higher than 1.5 M markedly inhibited cell growth, increase of initial NaCl concentration from 0.5 (equal to sea water) to 1.0 M resulted in a higher intracellular lipid content (67%) in comparison with 60% for the salt concentration of 0.5 M. Addition of 0.5 or 1.0 M NaCl at mid-log phase or the end of log phase during cultivation with initial NaCl concentration of 1.0 M further increased the lipid content (70%).

Tendon xanthomas in familial hypercholesterolemia are associated with a differential inflammatory response of macrophages to oxidized LDL

Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX−), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX− were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX− have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real-time RT-PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF-α, IL-8 and IL-6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.

Addition of Sodium Chlorine on culture of Dunaliella tertiolecta ATCC 30929 :Implication on Intracellular Lipid Content

The effect of addition sodium chloride (NaC1) on the accumulation intracellular of lipid in Dunaliella cells was investigated. Although initial NaC1 concentration higher than 1.5 M markedly inhibited cell growth, increase of initial NaCI concentration from 0.5 (eq. to sea water) to 1.0 M resulted in a higher intracellular lipid content (67%) in comparison with 60% for the salt concentration of 0.5 M Addition of 0.5 or 1.0 M NaC1 at mid-log phase or the end of log phase during culture with initial NaC1 concentration of 1.0 M further increased the lipid content (70%). Addition of 2.0 M NaC1 decreased final cell concentration to almost half of that without addition, although it resulted in very high lipid content of 77%.

The interplay among cardiac ultrastructure, metabolism and the expression of oxygen-binding proteins in Antarctic fishes.

We examined heart ventricle from three species of Antarctic fishes that vary in their expression of oxygen-binding proteins to investigate how some of these fishes maintain cardiac function despite the loss of hemoglobin (Hb) and/or myoglobin (Mb). We quantified ultrastructural features and enzymatic indices of metabolic capacity in cardiac muscle from Gobionotothen gibberifrons, which expresses both Hb and Mb, Chionodraco rastrospinosus, which lacks Hb but expresses Mb, and Chaenocephalus aceratus, which lacks both Hb and Mb. The most striking difference in cellular architecture of the heart among these species is the percentage of cell volume occupied by mitochondria, V(v)(mit,f), which is greatest in Chaenocephalus aceratus (36.53+/-2.07), intermediate in Chionodraco rastrospinosus (20.10+/-0.74) and lowest in G. gibberifrons (15.87+/-0.74). There are also differences in mitochondrial morphologies among the three species. The surface area of inner mitochondrial membrane per volume of mitochondria, S(v)(imm, mit), varies inversely with mitochondrial volume density so that S(v)(imm,mit) is greatest in G. gibberifrons (29.63+/-1.62 microm(-)(1)), lower in Chionodraco rastrospinosus (21.52+/-0.69 microm(-)(1)) and smallest in Chaenocephalus aceratus (20.04+/-0.79 microm(-)(1)). The surface area of mitochondrial cristae per gram of tissue, however, is greater in Chaenocephalus aceratus than in G. gibberifrons and Chionodraco rastrospinosus, whose surface areas are similar. Despite significant ultrastructural differences, oxidative capacities, estimated from measurements of maximal activities per gram of tissue of enzymes from aerobic metabolic pathways, are similar among the three species. The combination of ultrastructural and enzymatic data indicates that there are differences in the density of electron transport chain proteins within the inner mitochondrial membrane; proteins are less densely packed within the cristae of hearts from Chaenocephalus aceratus than in the other two species. High mitochondrial densities within hearts from species that lack oxygen-binding proteins may help maintain oxygen flux by decreasing the diffusion distance between the ventricular lumen and mitochondrial membrane. Also, high mitochondrial densities result in a high intracellular lipid content, which may enhance oxygen diffusion because of the higher solubility of oxygen in lipid compared with cytoplasm. These results indicate that features of cardiac myocyte architecture in species lacking oxygen-binding proteins may maintain oxygen flux, ensuring that aerobic metabolic capacity is not diminished and that cardiac function is maintained.

Are Water Permeability Measurements Sufficient to Characterize in Vitro Cultured Human Skin Surrogates?

We have found previously that human neonatal foreskin keratinocyte air/liquid (A/L) cultures developed normal-appearing epidermis, and that water flux was only two to three times higher than that found with intact human skin. To understand further the barrier properties of these A/L cultures, we have analyzed the composition and the gross conformational structures of the cultured stratum corneum (SC) lipids, and compared them with those of human SC. Electron microscopic examination demonstrated that cultured SC has high intracellular lipid content, but that it lacks both the basic unit repetition (e.g., broad/narrow/broad/broad/narrow/ broad pattern of electronlucent bands) normally found in human SC intercellular lipids and the covalently bound lipid envelope. These results indicate that the cultures are hyperproliferative, with characteristics similar to those found in SC obtained from patients with atopic dermatitis or psoriasis, or from animals whose diets lack essential fatty acids. In addition, the high free sterol content and the altered fatty acid/ceramide composition of these cultures argue that the compromised barrier function is linked to hyperproliferation and lipid synthesis, or vice versa. Infrared spectroscopic analysis of the SC lipid thermal transitions confirm that there are major conformational differences between the lipids of cultured and human SC, which clearly have an impact on permeability. The hyperproliferative state of growth and the profound differences between cultured and human SC in their lipid structural, compositional, and conformational properties together attest that water permeability alone is not a sufficiently sensitive marker of keratinocyte terminal differentiation for in vitro culture systems. It is not completely obvious how these differences in the cultured SC may influence the utility of this surrogate in studies involving skin biochemistry, pharmacology, permeability, metabolism, and toxicology. Nevertheless, it is clear that one needs to characterize the end product fully before one can judiciously interpret any results obtained from this or other similar skin surrogates.

Red and White Fiber Content and Associated Post‐Mortem Properties of Seven Porcine Muscles

SUMMARYThe red fiber content of several porcine muscles was estimated by a histochemical method (affinity for Sudan Black B due to high intracellular lipid content) and a biochemical method (high succinic dehydrogenase activity). Even though the muscles varied widely in total lipid content, the histochemical method was comparable to the biochemical method in categorizing porcine muscles as red or white. Red muscles (> 40% red fibers) contained greater myoglobin concentrations and generally had longer post‐rigor sarcomeres than white (< 30% red fibers) muscles. Interrelationships of percent red fibers, succinic dehydrogenase activity, fat content, glycolytic rate, and post‐rigor contraction state were discussed.