Neutral Lipid Content Research Articles

Interactions between cortisol and lipids in human milk

BackgroundHuman breast milk is one of the key early postnatal biological exposures for the developing child. It includes bioactive compounds, such as cortisol and fatty acids, which may be linked via the mother’s lipid metabolism.MethodsThis study investigated the associations between cortisol and lipids in human milk at the infant age of 2.5 months. Human milk cortisol concentrations were measured using luminescence immunoassay, and two groups of milks (n = 50 each) were formed based on either high (> 10 nmol/L) or low (< 3 nmol/L) cortisol levels. Lipids, as fatty acid content and composition of neutral (triacylglycerol-rich) and polar (phospholipid-rich) lipids, were measured with gas chromatography. The samples originated from the FinnBrain Birth Cohort Study.ResultsThe percentage of phospholipid-rich lipids of total lipids was 33.08% ± 1.33%. In triacylglycerol-rich lipids, high cortisol level in milk was associated with higher lauric (12:0, mass % and mg/mL), myristic (14:0, mass % and mg/mL), eicosenoic (20:1n − 9, mass %), docosenoic (22:1n − 9, mass %, and mg/mL) acids, and to lower palmitic acid (16:0, mass %) compared with low cortisol levels in milk. In phospholipid-rich lipids, high cortisol level was associated with higher myristic (14:0, mass %) and docosenoic (22:1n − 9, mass %) acids. After adjusting for pre-pregnancy BMI and sampling time by linear regression, the milk cortisol remained a significant predictor for lauric and myristic acids in triacylglycerol-rich lipids, and myristic and docosenoic acid in phospholipid-rich lipids (β = 0.23 to 0.38 and p < 0.05 for each).ConclusionsThis study revealed certain significant associations between milk cortisol and the fatty acid composition of human milk, indicating that cortisol might be one of the factors affecting the origin of the lipids in human milk.

Starvation-induced lipid reserve depletion in Pratylenchus brachyurus leads to decreased infectivity in maize roots

Summary Nematode body neutral lipid (triacylglycerol) content has been related to infectivity and has direct implications in control strategies. In this study, Pratylenchus brachyurus populations were split into two groups: i) freshly hatched second-stage juveniles (J2) containing lipids stored during embryogenesis; ii) third- and fourth-stage juveniles (J3/J4) plus females that replenished their lipid reserves by feeding on maize (Zea mays) roots. These groups were subjected to starvation to study their lipid consumption dynamics by staining with Oil Red O, which binds specifically to neutral lipids. Before starvation, freshly hatched J2 had 27% of their body area stained, whereas J3/J4 and females had 75%. Freshly hatched J2 starved for 28 days at 25°C in water lost 63.8% of the original neutral lipid content, which caused a reduction of 91% of infectivity in maize roots. By contrast, J3/J4 and females exposed to the same conditions lost 56.7% of the original neutral lipid content, which resulted in less than 50% reduction in infectivity. During the period of food deprivation, J2 had a mean daily neutral lipid consumption rate of 0.63% and the other infectious stages (J3/J4 and females) had a mean daily neutral lipid consumption rate of 1.46% per day. This study adds information on the dynamics of lipid utilisation that supports the use of longer waiting periods for planting crops after fallow in soils infested with P. brachyurus as compared to Meloidogyne spp.-infested soils.

Enzymatic in-situ transesterification of neutral lipids from simulated wastewater cultured Chlorella emersonii and Pseudokirchneriella subcapitata to sustainably produce fatty acid methyl esters

Alternative, more sustainable and environmentally positive, sources of energy are one of the current global challenges. One approach to achieving more sustainable sources of energy is to use waste from one system as a raw material for energy production, following the circular biosystem philosophy. This study successfully adopted this approach whereby microalgae strains Chlorella emersonii and Pseudokirchneriella subcapitata were bioprospected and metabolically engineered in simulated wastewater supplemented with glucose to produce neutral lipids. Using a two-step cultivation approach neutral lipid content was enhanced in Chlorella emersonii and Pseudokirchneriella subcapitata biomass. Via in-situ transesterification, these neutral lipids were subsequently bioconverted to biodiesel feedstock fatty acid methyl esters using novel solvent stable lipase(s) from Pseudomonas reinekei and Pseudomonas brenneri. The culturing of appropriate microalgae on wastewater, and bioconversion via organo-stable lipases may provide a commercially viable and sustainable biodiesel feedstock to help address the current global energy challenge.

Therapeutic Targeting of the Secreted Lysophospholipase D Autotaxin Suppresses Tuberous Sclerosis Complex-Associated Tumorigenesis.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by multiorgan hamartomas, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). TSC2 deficiency leads to hyperactivation of mTOR Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Phospholipid metabolism is dysregulated upon TSC2 loss, causing enhanced production of lysophosphatidylcholine (LPC) species by TSC2-deficient tumor cells. LPC is the major substrate of the secreted lysophospholipase D autotaxin (ATX), which generates two bioactive lipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). We report here that ATX expression is upregulated in human renal angiomyolipoma-derived TSC2-deficient cells compared with TSC2 add-back cells. Inhibition of ATX via the clinically developed compound GLPG1690 suppressed TSC2-loss associated oncogenicity in vitro and in vivo and induced apoptosis in TSC2-deficient cells. GLPG1690 suppressed AKT and ERK1/2 signaling and profoundly impacted the transcriptome of these cells while inducing minor gene expression changes in TSC2 add-back cells. RNA-sequencing studies revealed transcriptomic signatures of LPA and S1P, suggesting an LPA/S1P-mediated reprogramming of the TSC lipidome. In addition, supplementation of LPA or S1P rescued proliferation and viability, neutral lipid content, and AKT or ERK1/2 signaling in human TSC2-deficient cells treated with GLPG1690. Importantly, TSC-associated renal angiomyolipomas have higher expression of LPA receptor 1 and S1P receptor 3 compared with normal kidney. These studies increase our understanding of TSC2-deficient cell metabolism, leading to novel potential therapeutic opportunities for TSC and LAM. SIGNIFICANCE: This study identifies activation of the ATX-LPA/S1P pathway as a novel mode of metabolic dysregulation upon TSC2 loss, highlighting critical roles for ATX in TSC2-deficient cell fitness and in TSC tumorigenesis.

Fatty Acid Characterization of Beef Longissimus from Steers Finished on Fodder Beet or Traditional Winter Forages in New Zealand Compared to US Grain-Fed Beef

Fatty acids were evaluated to characterize lipid profiles of beef from 3 feeding programs: New Zealand (NZ) fodder beet (FB), NZ grass/non-FB (NFB), and United States grain. Strip loins were selected within each program based on expected eating quality focusing on marbling, ossification, and pH. Selection resulted in 6 treatments: FB high quality, FB low quality, NFB high quality, NFB low quality, US Top Choice, and US Select. Samples were aged 21 d or 35 d postmortem. A subset of 152 samples (12–13 per treatment combination) were used for fatty acid characterization. Within the polar lipid fraction, US treatments had lower monounsaturated fatty acid (MUFA) and greater polyunsaturated fatty acid (PUFA) than NZ treatments (P &amp;lt; 0.05). Within the neutral lipid fraction, concentrations of total neutral lipids and proportions of saturated fatty acid, MUFA, and PUFA were affected by treatment (P &amp;lt; 0.05). Total neutral lipids were greatest in Top Choice (P &amp;lt; 0.05) and greater in FB high quality than all other treatments (P &amp;lt; 0.05). Proportions of satu- rated fatty acid in the neutral lipid fraction were greater in NFB treatments than US treatments (P &amp;lt; 0.05); FB treatments were intermediate. Proportions of MUFA were greatest in Top Choice (P &amp;lt; 0.05). Proportions of PUFA were greatest (P &amp;lt; 0.05) in Select, greater in Top Choice than all NZ treatments (P &amp;lt; 0.05), and greater in NFB treatments than FB treatments (P &amp;lt; 0.05). No interactions occurred for any groups of fatty acids in either fraction ( P &amp;gt; 0.05), and aging did not affect fatty acid composition. Lipid profiles were impacted by finishing diet; however, most differences were between US grain-fed beef and NZ treatments—with limited differences between FB and NFB samples—when product was selected based on quality descriptors. Therefore, FB could be substituted for grass forages to sustain the NZ beef industry through winter months and limit seasonality without detrimental effects on product quality.

315 Lipid accumulation in epidermal Langerhans cells correlate with aberrant immunofunctions in psoriasis-like inflammation

Psoriasis is a chronic, inflammatory, systemic disease characterized by abnormal lipid metabolism. Dendritic cells (DCs), an essential mediator in the pathogenesis of psoriasis, are recently discovered to be functionally regulated by intracellular lipid content. Being the sole DC subset within the epidermis, Langerhans cells (LCs) are potent regulators of immune surveillance and tolerance. Previous studies indicate a varying role of LCs in psoriasis with underpinning mechanism remains elusive. Here, we demonstrated aberrant maturation, enhanced phagocytosis and interleukin-23 (IL-23) over-secretion of LCs in imiquimod (IMQ)-induced psoriasis-like mouse skin. Remarkably, disease-associated LCs possessed an elevated level of neutral lipid content probably due to impaired autophagy of lipids other than altered lipid engulfment. Moreover, the inhibition of fatty acids synthesis, which resulted in a decrease in cellular neutral lipids, would significantly impair LC maturation and their production of IL-23. Further LC-MS analysis revealed a group of triglycerides (TG), diglycerides (DG), phosphatidylethanols (PEt) and phosphatidylcholines (PC) contributed to the increased neutral lipid level of LCs within IMQ-induced psoriasis-like skin. In accordance, low-input mRNA sequencing analysis uncovered the expressions of multiple genes involved in lipid metabolism, autophagy along with immunofunctions were correlatively dysregulated in murine disease-associated LCs. Single-cell RNA sequencing of psoriasis patients' LCs demonstrated an immune activation status of lesional LCs, which confirmed their pro-inflammatory role in the pathogenesis of psoriasis. Notably, hyperlipidemia does not directly influence LC homeostasis and immunofunction. In summary, lipid accumulation in epidermal Langerhans cells correlate with their aberrant immunofunctions in psoriasis-like inflammation.

Optimum interaction of light intensity and CO2 concentration in bioremediating N-rich real wastewater via assimilation into attached microalgal biomass as the feedstock for biodiesel production

Impoverishing nutrients from wastewater via assimilation into microalgal biomass has gained footprint in bioremediation technologies. To ease the harvesting of mature microalgal biomass from effluent, this study proposed the employment of fluidized bed bioreactor to grow attached microalgal biomass onto polyurethane foam support material while bioremediating the N-rich real wastewater from chemical fertilizer manufacturing industry. The complete removals of all nitrogen species (NH4+-N, NO2−-N and NO3−-N) together with COD and total phosphorus were achieved at the optimum light intensity and CO2 concentration of 216μmol/m2 s and 9.1 %, respectively, giving rise to the attached microalgal biomass productivity of 0.094g/L/day. These performances were also found maintaining for at least 4 cycles of reiterative uses of spent polyurethane foam support material in similar fluidized bed bioreactor setup condition with every cycle being introduced with fresh N-rich real wastewater. The mechanism of attachment formations study predicted that the polysaccharides and proteins from microalgal extracellular polymeric substances had bridged the cells onto polyurethane foam support material during the initial colonization prior to populating and growing to cover the surfaces of support material. For biodiesel production, the extracted neutral lipid content of attached microalgal biomass was found to be four times higher than the suspended growth culture. From the transesterification process, about 97 %–98 % of attached microalgal lipid could be converted into fatty acid methyl esters (FAMEs) mixture. Interestingly, the degree of saturated fatty acids (SFA) in FAMEs was recorded increasing with the increase of cycles of reusing spent polyurethane foam support material. The presence of more SFA in FAMEs could overall enhance the oxidative stability and contribute to higher cetane number of produced biodiesel from facilely harvested attached microalgal biomass.

Green-lipped mussel (Perna canaliculus) hemocytes: A flow cytometric study of sampling effects, sub-populations and immune-related functions

Green-lipped mussels (Perna canaliculus) are a commercially and culturally important bivalve species in New Zealand (NZ). As the highest value export aquaculture product in NZ, understanding and safeguarding the health of this species is imperative. The identification and characterization of hemocytes can provide useful information regarding the health of this species. Using flow cytometry (FCM), the present study assessed for the first time the use of different antiaggregant solutions and storage times on the immune-related parameters of hemocytes from cultured adult P. canaliculus. In addition, characterization of the immune-related functions of hemocyte sub-populations within the hemolymph were assessed. The two antiaggregant solutions tested (Modified Alserver's, MAS, A and B) maintained similar numbers of hemocytes in circulation over a 60 min period but, reduced the viability (MAS A) and increased the ROS production (MAS B) of the hemocytes compared to hemocytes diluted in cold filtered seawater (FSW). Hemocytes diluted in FSW and kept on ice showed significant aggregation after 2 h and a reduction in viability from 4 h. Three different hemocyte sub-populations were identified, discernible by their relative size and internal complexity: blast-like cells, hyalinocytes and granulocytes, which accounted for approximately 4, 67 and 29% of the total hemolymph population respectively. Granulocytes showed significantly higher reactive oxygen species production, phagocytic capabilities and neutral lipid content compared to hyalinocytes and blast-like cells. Results indicate that maintaining extracted hemolymph in cold FSW, completing analysis of fresh samples within 2 h of extraction and FCM assay incubation times of no longer than 30 min are best to obtain accurate results. Formalin fixation can also be used for future determination of hemocyte sub-populations and internal structures. Results from this study will allow effective future study of the effects of various stressors on P. canaliculus health and lead to improved management and production strategies in this species.

SAT-588 Role of Adipose Tissue in Reproduction, Mammary Gland Development, Function and Cancer

To investigate the role of adipose tissue in the function of the mammary gland (MG) and reproductive system, we have examined lipodystrophic (LD) mice. LD mice of both sexes are sterile, but fertility was restored in both sexes with leptin injections. In addition, leptin was only needed for initial stages of pregnancy and not for parturition. A transplant of mouse embryonic fibroblasts (MEFs) led to the formation of an ectopic fat pad which also rescued the fertility in both sexes. However, pups born to rescued LD mothers died shortly after birth. We therefore examined the mammary glands of these mothers. MGs from LD mice were rudimentary and lacked terminal end buds. Leptin-injected and MEF rescued LD mice were able to become pregnant, showed normal pregnancy-associated glandular proliferation despite a smaller glandular area, were able to produce a small amount of milk that had grossly normal content of milk proteins and neutral lipids, but could not sustain pups to weaning. In order to separate the individual requirements for 1) adipokines such as leptin, 2) estradiol, and 3) epithelial-adipocyte interactions, we performed a series of experiments with both LD and ob (leptin-deficient) mice that received either estradiol or preadipocyte transplant. The resulting fat pad did not rescue the defect in MG development in LD mice. The defect also was not rescued with estradiol pellets. Ob/ob mice, like LD mice, lack leptin and estradiol, but retain adipose tissue. Ob mice have defective MG development. However, in striking contrast to LD mice, reconstitution of a WT fat pad in ob mice rescued the defect in MG development. Estradiol treatment did not rescue MG development in ob mice. Therefore direct interaction between mammary gland epithelia and adipocytes is a requirement for full invasion and expansion of the gland during puberty, but is not required for glandular proliferation during pregnancy and milk production.Given that excess adipose tissue is a risk factor for breast cancer we wanted to determine if breast cancer was affected by the absence of adipose tissue. LD mice were bred to MMTV-PyMT mice that develop spontaneous breast cancer. Remarkably, LD PyMT+ mice had accelerated growth of primary tumors compared to WT PyMT+ mice. Using our MEF transplant model future studies will be directed to understanding whether the accelerated breast cancer growth is due to loss of adipokines or altered epithelial-stromal interactions.

Automated Pressurized Liquid Extraction of Microbial Lipids from Oleaginous Yeasts

The lipids produced by oleaginous microbes are considered sustainable resources for biofuels. To facilitate controlled lipid production and lipid analysis, more efficient lipid extraction methods are required. This study describes the automated pressurized liquid extraction (APLE) method for lipid extraction from dried cells of the oleaginous yeast species Rhodosporidium toruloides and Cryptococcus curvatus. Cells were mixed with diatomite in a mortar, added to the sample chamber, and treated with a mixture of chloroform and methanol at 105°C. More than 95% lipids were extracted. Analysis by using high-performance thin-layer chromatography showed that the neutral lipid contents in the obtained samples by APLE method were similar to those by the ball milling-assisted extraction method. The lipids had an essentially identical fatty acid composition compared with lipids extracted with the acid-heating extraction (AHE) method. This demonstrated that lipids can be efficiently extracted from oleaginous yeasts in less time and without harsh pretreatment procedures.

Toxicity of the cosmetic preservatives parabens, phenoxyethanol and chlorphenesin on human meibomian gland epithelial cells

Recently, we discovered that the cosmetic preservatives, benzalkonium chloride and formaldehyde, are especially toxic to human meibomian gland epithelial cells (HMGECs). Exposure to these agents, at concentrations approved for human use, leads within hours to cellular atrophy and death. We hypothesize that these effects are not unique, and that other cosmetic preservatives also exert adverse effects on HMGECs. Such compounds include parabens, phenoxyethanol and chlorphenesin, which have been reported to be toxic to corneal and conjunctival epithelial cells, the liver and kidney, as well as to irritate the eye. To test our hypothesis, we examined the influence of parabens, phenoxyethanol and chlorphenesin on the morphology, signaling, survival, proliferation and lipid expression of immortalized (I) HMGECs. These cells were cultured under proliferating or differentiating conditions with varying concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin for up to 5 days. We monitored the signaling ability, appearance, number and neutral lipid content of the IHMGECs, as well as their lysosome accumulation. Our findings show that a 30-min exposure of IHMGECs to these preservatives results in a significant reduction in the activity of the Akt pathway. This effect is dose-dependent and occurs at concentrations equal to (chlorphenesin) and less than (all others) those dosages approved for human use. Further, a 24-h treatment of the IHMGECs with concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin close to, or at, the approved human dose induces cellular atrophy and death. At all concentrations tested, no preservative stimulated IHMGEC proliferation. Of particular interest, it was not possible to evaluate the influence of these preservatives, at close to human approved dosages, on IHMGEC differentiation, because the cells did not survive the treatment. In summary, our results support our hypothesis and show that methylparaben, ethylparaben, phenoxyethanol and chlorphenesin are toxic to IHMGECs.

The Role of Hypoxia-Inducible Factor 1α in the Regulation of Human Meibomian Gland Epithelial Cells.

PurposeWe recently discovered that a hypoxic environment is beneficial for meibomian gland (MG) function. The mechanisms underlying this effect are unknown, but we hypothesize that it is due to an increase in the levels of hypoxia-inducible factor 1α (HIF1α). In other tissues, HIF1α is the primary regulator of cellular responses to hypoxia, and HIF1α expression can be induced by multiple stimuli, including hypoxia and hypoxia-mimetic agents. The objective of this study was to test our hypothesis.MethodsHuman eyelid tissues were stained for HIF1α. Immortalized human MG epithelial cells (IHMGECs) were cultured for varying time periods under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the hypoxia-mimetic agent roxadustat (Roxa). IHMGECs were then processed for the analysis of cell number, HIF1α expression, lipid-containing vesicles, neutral and polar lipid content, DNase II activity, and intracellular pH.ResultsOur results show that HIF1α protein is present in human MG acinar epithelial cells in vivo. Our findings also demonstrate that exposure to 1% O2 or to Roxa increases the expression of HIF1α, the number of lipid-containing vesicles, the content of neutral lipids, and the activity of DNase II and decreases the pH in IHMGECs in vitro.ConclusionsOur data support our hypothesis that the beneficial effect of hypoxia on the MG is mediated through an increased expression of HIF1α.

Microalgal swimming signatures and neutral lipids production across growth phases.

Microalgae have been shown as a potential bioresource for food, biofuel, and pharmaceutical products. During the growth phases with corresponding environmental conditions, microalgae accumulate different amounts of various metabolites. We quantified the neutral lipids accumulation and analyzed the swimming signatures (speed and trajectories) of the motile green alga, Dunaliella primolecta, during the lag-exponential-stationary growth cycle at different nutrient concentrations. We discovered significant changes in the neutral lipid content and swimming signatures of microalgae across growth phases. The timing of the maximum swimming speed coincided with the maximum neutral lipid content and both maxima occurred under nutrient stress at the stationary growth phase. Furthermore, the swimming trajectories suggested statistically significant changes in swimming modes at the stationary growth phase when the maximum intracellular neutral lipid content was observed. Our results provide the potential exploitation of microalgal swimming signatures as possible indicators of the cultivation conditions and the timing of microalgal harvest to maximize the lipid yield for biofuel production. The findings can also be implemented to explore the production of food and antibiotics from other microalgal metabolites with low energy costs.

Live-Cell Superresolution Microscopy of Faa4 Re-Distribution on Lipid Droplets during Metabolic Transitions in Yeast

Lipid droplets (LDs) are energy storage organelles of a cell with a neutral lipid core surrounded by a phospholipid monolayer. Cells achieve their lipid and energy homeostasis by actively regulating the synthesis and the breakdown of neutral lipids stored in LDs during metabolic transitions. In order to accommodate the change in size, number and neutral lipid content of LDs, cells regulate the abundance and the density of specific proteins involved in the synthesis and the breakdown of neutral lipids. In yeast, Faa4 encodes for one of the fatty acyl-CoA synthetases required for the activation of fatty acids essential for making neutral lipids. In this study, we employ correlative Photo-Activated Localization Microscopy (PALM) and conventional fluorescence microscopy to resolve and quantify the density of Faa4 on LDs below the optical diffraction limit, on the ER, and on the vacuole as yeast cells transition between exponential, stationary and lag phases based on the nutrient availability. We find that the Faa4 density on LDs increases as LDs grow in size and re-localize to the vacuolar membrane from ER when the cell progresses to the stationary phase from the exponential growth phase. However, Faa4 is predominately localized to vacuolar lumen within ∼2 hrs of dilution in fresh media suggesting rapid consumption of LDs upon growth resumption. The increase in Faa4 density on LDs that are about to enter the vacuole for breakdown may therefore be the major mechanism how Faa4 shuttles into the vacuole to activate fatty acids. Our approach to resolve and quantify the density of regulatory proteins on LDs in living cells may help in future studies to better understand how cells differentially regulate their density to maintain lipid and energy homeostasis based on the metabolic needs.

Early biomarker indicators of health in two commercially produced microalgal species important for aquaculture

In bivalve hatcheries, microalgae production accounts for a large proportion of hatchery operation costs. A reliable supply of good quality algae is essential for optimal output. Two commercially important algal species, Tisochrysis lutea (T-Iso) and Chaetoceros calcitrans were grown in batch culture under optimal and high-pH conditions for 7 and 5 days respectively. A suite of parameters were compared to identify those that could be considered as early markers of algal stress. One day after inoculation of both algal species, carbon dioxide (CO2) addition was removed to cause the high pH treatment condition and cultures were monitored to determine any changes in algal health. A variety of indicators of algal function including photosynthetic parameters by Pulse Amplitude Modulation (PAM) fluorometry and morphological and functional changes by flow cytometry were determined in parallel during the pH stress to correlate them and identify parameters that could be used as an early indicator of decreasing algal health. The pH in the sub-optimal treatments was significantly higher compared with controls from 1 day after the removal of CO2 addition. At the same sampling time (Day 2), in the high-pH treatment, the PSII photosynthetic efficiency in the light measured by PAM fluorometry and, chlorophyll fluorescence and reactive oxygen species production measured by flow cytometry were significantly lower for T-Iso, compared with controls. In sub-optimal cultures of C. calcitrans, relative algal size, complexity, chlorophyll and neutral lipid content (as measured by flow cytometry) were higher as early as Day 2, compared with controls. Additionally, the bacterial abundance associated with T-Iso and C. calcitrans cells was increased in the high pH treatment compared with controls, from Day 6 and Day 5 respectively. These responses to a subtle negative change in the culture environment were detected 24 h prior to a change in cell density and indicate the promise of PAM fluorometry and flow cytometry as tools to provide a rapid, sensitive and reliable assessment of microalgal health in a hatchery setting. Such advances in algal health monitoring will improve bivalve production systems.

The Marine Seagrass Halophila stipulacea as a Source of Bioactive Metabolites against Obesity and Biofouling.

Marine organisms, including seagrasses, are important sources of biologically active molecules for the treatment of human diseases. In this study, organic extracts of the marine seagrass Halophila stipulacea obtained by different polarities from leaves (L) and stems (S) (hexane [HL, HS], ethyl acetate [EL, ES], and methanol [ML, MS]) were tested for different bioactivities. The screening comprehended the cytotoxicity activity against cancer cell lines grown as a monolayer culture or as multicellular spheroids (cancer), glucose uptake in cells (diabetes), reduction of lipid content in fatty acid-overloaded liver cells (steatosis), and lipid-reducing activity in zebrafish larvae (obesity), as well as the antifouling activity against marine bacteria (microfouling) and mussel larval settlement (macrofouling). HL, EL, HS, and ES extracts showed statistically significant cytotoxicity against cancer cell lines. The extracts did not have any significant effect on glucose uptake and on the reduction of lipids in liver cells. The EL and ML extracts reduced neutral lipid contents on the larvae of zebrafish with EC50 values of 2.2 µg/mL for EL and 1.2 µg/mL for ML. For the antifouling activity, the HS and ML extracts showed a significant inhibitory effect (p < 0.05) against the settlement of Mytilus galloprovincialis plantigrade larvae. The metabolite profiling using HR-LC-MS/MS and GNPS (The Global Natural Product Social Molecular Networking) analyses identified a variety of known primary and secondary metabolites in the extracts, along with some unreported molecules. Various compounds were detected with known activities on cancer (polyphenols: Luteolin, apeginin, matairesinol), on metabolic diseases (polyphenols: cirsimarin, spiraeoside, 2,4-dihydroxyheptadec-16-ynyl acetate; amino acids: N-acetyl-L-tyrosine), or on antifouling (fatty acids: 13-decosenamide; cinnamic acids: 3-hydroxy-4-methoxycinnamic acid, alpha-cyano-4-hydroxycinnamic), which could be, in part, responsible for the observed bioactivities. In summary, this study revealed that Halophila stipulacea is a rich source of metabolites with promising activities against obesity and biofouling and suggests that this seagrass could be useful for drug discovery in the future.

Myeloid atg5 deletion impairs n-3 PUFA-mediated atheroprotection

Background and aimsDietary long-chain (≥20 carbons) n-3 polyunsaturated fatty acids (PUFAs) reduce atherosclerosis and enhance macrophage autophagy activation. How macrophage autophagy impacts atherosclerotic progression, particularly when comparing dietary n-3 PUFA supplementation vs. saturated fat feeding, is unknown. MethodsWe generated myeloid-specific autophagy-deficient and control mice in the Ldlr−/− background by transplanting bone marrow from myeloid-specific autophagy-related (atg) 5 knockout mice and wild type controls into irradiated Ldlr−/− recipients. After 7 weeks for recovery from radiation, mice were fed an atherogenic diet containing 0.2% cholesterol and 20% calories as palm oil (PO diet), or 10% calories as PO plus 10% calories as fish oil (FO diet) for 16 weeks. ResultsCompared to PO, FO significantly reduced plasma cholesterol, triglyceride, hepatic neutral lipid, and aortic caspase-1 cleavage, but increased aortic efferocytosis, leading to attenuated atherosclerosis in Ldlr−/− mice receiving wild type bone marrow. Myeloid atg5 deletion had little impact on plasma lipid concentrations and hepatic neutral lipid content, regardless of diet. Myeloid atg5 deletion increased aortic caspase-1 cleavage, decreased aortic efferocytosis and worsened atherosclerosis only in the FO-fed Ldlr−/− mice. ConclusionsDeficient myeloid autophagy significantly attenuated FO-induced atheroprotection, suggesting that dietary n-3 PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy.

Chemical characteristics of peripheral blood granulocyte biomembranes in frequently ill children with autonomic dysfunction syndrome

The problem of frequently ill children has extreme relevance in medical practice. A special group is FIC with autonomic dysfunction syndrome, born prematurely with perinatal damage of the central nervous system (CNS). In previous studies, we found that FIC who were born prematurely with perinatal damage of the central nervous system exhibit lower immunoreactivity than their healthy peers. Purpose of the work – to study the biochemical indicator of immunocompetent cells (the study of the phospholipid composition of granulocyte membranes) of the frequently ill children with autonomic dysfunction syndrome, born prematurely with perinatal damage to the central nervous system (CNS). Materials and methods. 68 children in the age of 6-7 years who are belonging to the group of frequently ill were examined. Out of 68 children, there were 36 children with autonomic dysfunction syndrome (SVD) born prematurely with perinatal hypoxic-ischemic damage to the central nervous system (group 1) and 32 children without autonomic dysfunction syndrome born on time without central nervous system damage (group 2). Results. A study of the phospholipid composition of blood granulocyte membranes of patients of the studied groups revealed significant differences comparison with the control group. The revealed changes in granulocyte biomembranes were more pronounced for FIC with SVD born prematurely with perinatal CNS damage than for children born on time without CNS and SVD damage. Conclusions. The obtained data allow us to conclude that FIL who were born preterm with perinatal damage of the central nervous system, changes in the biochemical characteristics of membranes are observed to a slightly greater extent than for children who were born on time without central nervous system damage. The revealed changes in immunocompetent cells are characterized by an increase in the polarity of the lipid component of biomembranes and a decrease in the relative content of neutral lipids in them. These violations lead to a change in protein-lipid interactions, which, as a result, leads to a change in the microviscosity of biomembranes and their structural and functional properties. At these children forme an inferior immune response to an infectious agent.

Bioactivity-Guided Isolation and Identification of Anti-adipogenic Constituents from the n-Butanol Fraction of Cissus quadrangularis.

Obesity is marked by the buildup of fat in adipose tissue that increases body weight and the risk of many associated health problems, including diabetes and cardiovascular disease. Treatment options for obesity are limited, and available medications have many side effects. Thus there is a great need to find alternative medicines for treating obesity. This study explores the anti-adipogenic potential of the n-butanol fraction of Cissus quadrangularis (CQ-B) on 3T3-L1 mouse preadipocyte cell line. The expression of various lipogenic marker genes such as adiponectin, peroxisome proliferator-activated receptor gamma, leptin, fatty acid-binding proteins, sterol regulatory element-binding proteins, fetal alcohol syndrome, steroyl-CoA desaturase-1, lipoproteins, acetyl-CoA carboxylase alpha, and acetyl-CoA carboxylase beta were variously significantly downregulated. After establishing the anti-adipogenic potential of CQ-B, it was fractionated to isolate anti-adipogenic compounds. We observed significant reduction in neutral lipid content of differentiated cells treated with various fractions of CQ-B. Gas chromatography-mass spectrometry analysis revealed the presence of thirteen compounds with reported anti-adipogenic activities. Further studies to purify these compounds can offer efficacious and viable treatment options for obesity and related complications.

Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.