Umbilical Cord Plasma Lysophospholipids and Triacylglycerols Associated with Birthweight Percentiles.

Dysregulated transplacental lipid transfer and fetal-placental lipid metabolism affect birthweight, as does maternal hyperglycemia. As the mechanisms are unclear, we aimed to identify the lipids in umbilical cord plasma that were most associated with birthweight. Seventy-five Chinese women with singleton pregnancies recruited into the GUSTO mother-offspring cohort were selected from across the glycemic range based on a mid-gestation 75 g oral glucose tolerance test, excluding pre-existing diabetes. Cord plasma samples collected at term delivery were analyzed using targeted liquid-chromatography tandem mass-spectrometry to determine the concentrations of 404 lipid species across 17 lipid classes. The birthweights were standardized for sex and gestational age by local references, and regression analyses were adjusted for the maternal age, BMI, parity, mode of delivery, insulin treatment, and fasting/2 h glucose, with a false discovery-corrected p < 0.05 considered significant. Ten lysophosphatidylcholines (LPCs) and two lysophosphatidylethanolamines were positively associated with the birthweight percentiles, while twenty-four triacylglycerols were negatively associated with the birthweight percentiles. The topmost associated lipid was LPC 20:2 [21.28 (95%CI 12.70, 29.87) percentile increase in the standardized birthweight with each SD-unit increase in log10-transformed concentration]. Within these same regression models, maternal glycemia did not significantly associate with the birthweight percentiles. Specific fetal circulating lysophospholipids and triacylglycerols associate with birthweight independently of maternal glycemia, but a causal relationship remains to be established.

Randomized, double-blind, placebo-controlled trial to examine the safety, pharmacokinetics and effects of Epimedium prenylflavonoids, on bone specific alkaline phosphatase and the osteoclast adaptor protein TRAF6 in post-menopausal women.

BACKGROUND: Fragility fractures due to menopausal osteoporosis are a major cause of morbidity and mortality. Osteoporotic medications have substantial side effects that limit long term use. HYPOTHESES: Ingestion of a purified extract of Epimedium spp. (EP) is safe, can increase serum levels of prenylflavonoid metabolites, exert positive changes in bone specific alkaline phosphatase (BSAP), suppress of tumor necrosis factor receptor associated factor 6 (TRAF6) protein in osteoclast-precursor monocytes in peripheral blood and therefore have the potential to reduce post-menopausal bone loss. STUDY DESIGN & METHODS: Healthy postmenopausal women were randomized in a double-blind fashion to consume either EP prenylflavonoid extract (740 mg daily) or placebo daily for 6 weeks. The main outcome measures were safety and pharmacokinetics of EP flavonoids. Fasting blood was collected at 3- and 6-weeks, and two weeks after stopping medication for safety evaluations and measurement of BSAP. Peripheral blood monocytes were harvested for measurement of TRAF6 levels. Serum levels of the EP metabolites icariin, icariside I & II, icaritin and desmethylicaritin were measured using tandem mass spectrometry, and non-compartmental pharmacokinetic analyses performed using WinNonlin software. RESULTS: Between October 2018 and Jun 2020, 58 postmenopausal women, aged 57.9 ± 8.9 years, were randomized and completed the study. Consumption of EP prenylflavonoids was not associated with any significant adverse symptoms, with no changes in hepatic, hematological, and renal parameters observed. The main metabolites detected in sera after ingestion of EP prenylflavonoid capsules were desmethylicaritin, icaritin and icariside II. Icariin and icariside I were below detection levels. Ingestion of EP prenylflavonoids induced a median Cmax and AUC0→∞ for desmethylicaritin of 60.9 nM, and 157.9 nM ×day, respectively; and were associated with higher levels of BSAP (p < 0.05) and a trend (p = 0.068) towards lower levels of TRAF6 in peripheral blood monocytes eight weeks after commencing prenylflavonoid ingestion. Prenylflavonoid metabolites were not detected in the sera of placebo participants. CONCLUSIONS: Despite the widespread consumption of EP extracts, the safety, mechanisms of action of their bioactive compounds, and therapeutic indications in humans are unknown. Daily consumption of EP prenylflavonoids for six weeks was safe. The predominant metabolite in sera was desmethylicaritin. Rise in prenylflavonoid metabolites was associated with higher levels of the bone anabolic marker BSAP, suggesting potential therapeutic value for post-menopausal osteoporosis.

Predictors of circulating vitamin D levels in healthy mid-life Singaporean women.

Vitamin D levels were lowest in Indian and Malay compared to Chinese women, and in younger and employed women. The main reason for hypovitaminosis D in study women was deficient cutaneous production. Supplementation in regions with abundant sunshine should consider ethnicity and opportunities for exposure to sunlight. INTRODUCTION: Little is known about variations in circulating vitamin D levels in multiethnic mid-life women living in communities with year-round sunlight. Our study describes the circulating vitamin D levels and their sociodemographic predictors in mid-life Singaporean women. METHODS: Prospective cross-sectional study of healthy Singaporean women, age 45-69 years, who were not consuming vitamin D supplements. Total 25-hydroxyvitaminD [25(OH)D], the sum of 25(OH)D2 and 25(OH)D3, was measured by liquid chromatography-tandem mass spectrometry. RESULTS: The analytic cohort of 721 women, mean age 55.2±6.0 (±SD) years, was of Chinese (82%), Indian (11%), and Malay (7%) ethnicity. Their mean 25(OH)D level was 24.8±7.8ng/mL. One-third (32.6%) of the women had deficient 25(OH)D (≤20ng/mL) and 3.5% were severely deficient (<12ng/mL). 25(OH)D3 comprised 98% of the total circulating 25(OH)D level. Adjusted mean total 25(OH)D levels were significantly lower for women of Indian and Malay (vs Chinese) ethnicity, who were premenopausal or working outside the home. Indian and Malay women had higher odds (adjusted OR 5.58 (95% CI 3.22, 9.87) and 3.83 (95% CI 1.97, 7.57), respectively) of low 25(OH)D compared to Chinese women. Obesity was not an independent predictor of low 25(OH)D, as its strong crude association was confounded by ethnicity. The adjusted odds of low 25(OH)D was reduced in women ≥65 years (adjusted OR 0.37 (95% CI 0.14, 0.87)) compared to those aged 45-55 years. CONCLUSION: One-third of mid-life Singaporean women were 25(OH)D deficient, and the major independent predictors of deficiency were Indian or Malay ethnicity and younger age. Vitamin D supplementation in mid-life women should be targeted to those with documented deficiency or limited cutaneous production.

Pharmacokinetics of Prenylflavonoids following Oral Ingestion of Standardized Epimedium Extract in Humans.

Leaves of the Epimedium plant are traditionally consumed for bone health and other indications. The aim of this study was to establish the safety and pharmacokinetics of the metabolites of prenylflavonoids (icariin, icariside I, icariside II, icaritin, and desmethylicaritin) following single doses of a defined Epimedium prenylflavonoid extract in humans. A single oral dose of 370, 740, or 1110 mg of a standardized Epimedium prenylflavonoid extract was administered to 30 healthy male subjects in a randomized, placebo-controlled trial. Serum samples were collected over a 48-h period and analyzed by liquid chromatography-tandem mass spectrometry and non-compartmental pharmacokinetic modelling. Epimedium prenylflavonoid extracts were well tolerated and no adverse effects were observed. The principle metabolites detected in the serum were icariside II and desmethylicaritin. Icariside II had a T max of between 4.1 - 4.3 h, reaching a maximum AUC0→∞ of 23.0 (17.5, 29.9) h×ng/mL (median [IQR: interquartile range]) with the highest dose of the Epimedium prenylflavonoid. On the other hand, desmethylicaritin had a delayed T max of 24.1 - 24.4 h and reached a maximum AUC0→∞ of 126.1 (62.4, 202.9) h×ng/mL. The median maximum plasma concentration and AUC0→∞ of desmethyliciaritin showed an increase with higher doses of the Epimedium prenylflavonoid (p < 0.05). Icariin, icariside I, and icaritin levels were below detection limits. Levels of Epimedium prenylflavonoid metabolites observed in this study were consistent with levels demonstrated to have anti-osteoporotic effects in cellular and animal studies. Coupled with the favorable safety profile of the extract observed, further studies are required to explore the utility of Epimedium prenylflavonoid extracts to prevent osteoporosis in postmenopausal women.

Sex Differences in 20-Hydroxyecdysone Hormone Levels Control Sexual Dimorphism in Bicyclus anynana Wing Patterns.

In contrast to the important role of hormones in the development of sexual traits in vertebrates (Cox RM, Stenquist DS, Calsbeek R. 2009. Testosterone, growth and the evolution of sexual size dimorphism. J Evol Biol. 22(8):1586-1598.), the differentiation of these traits in insects is attributed almost exclusively to cell-autonomous mechanisms controlled by members of the sex determination pathway (Verhulst EC, van de Zande L. 2015. Double nexus - doublesex is the connecting element in sex determination. Brief Funct Genomics 14(6):396-406.), such as doublesex. Although hormones can shape the development of sexual traits in insects, variation in hormone levels are not conclusively known to cause dimorphism in these traits (Prakash A, Monteiro A. 2016. Molecular mechanisms of secondary sexual trait development in insects. Curr Opin Insect Sci. 17:40-48.). Here, we show that butterflies use sex-specific differences in 20-hydroxyecdysone hormone titers to create sexually dimorphic wing ornaments. Females of the dry season (DS) form of Bicyclus anynana display a larger sexual ornament on their wings than males, whereas in the wet season form both sexes have similarly sized ornaments (Prudic KL, Jeon C, Cao H, Monteiro A. 2011. Developmental plasticity in sexual roles of butterfly species drives mutual sexual ornamentation. Science 331(6013):73-75.). High levels of circulating 20-hydroxyecdysone during larval development in DS females and wet season forms cause proliferation of the cells fated to give rise to this wing ornament, and results in sexual dimorphism in the DS forms. This study advances our understanding of how the environment regulates sex-specific patterns of plasticity of sexual ornaments and conclusively shows that hormones can play a role in the development of secondary sexual traits in insects, just like they do in vertebrates.

Curcumin Ameliorates Neuroinflammation, Neurodegeneration, and Memory Deficits in p25 Transgenic Mouse Model that Bears Hallmarks of Alzheimer's Disease.

Several studies have indicated that neuroinflammation is indeed associated with neurodegenerative disease pathology. However, failures of recent clinical trials of anti-inflammatory agents in neurodegenerative disorders have emphasized the need to better understand the complexity of the neuroinflammatory process in order to unravel its link with neurodegeneration. Deregulation of Cyclin-dependent kinase 5 (Cdk5) activity by production of its hyperactivator p25 is involved in the formation of tau and amyloid pathology reminiscent of Alzheimer's disease (AD). Recent studies show an association between p25/Cdk5 hyperactivation and robust neuroinflammation. In addition, we recently reported the novel link between the p25/Cdk5 hyperactivation-induced inflammatory responses and neurodegenerative changes using a transgenic mouse that overexpresses p25 (p25Tg). In this study, we aimed to understand the effects of early intervention with a potent natural anti-inflammatory agent, curcumin, on p25-mediated neuroinflammation and the progression of neurodegeneration in p25Tg mice. The results from this study showed that curcumin effectively counteracted the p25-mediated glial activation and pro-inflammatory chemokines/cytokines production in p25Tg mice. Moreover, this curcumin-mediated suppression of neuroinflammation reduced the progression of p25-induced tau/amyloid pathology and in turn ameliorated the p25-induced cognitive impairments. It is widely acknowledged that to treat AD, one must target the early-stage of pathological changes to protect neurons from irreversible damage. In line with this, our results demonstrated that early intervention of inflammation could reduce the progression of AD-like pathological outcomes. Moreover, our data provide a rationale for the potential use of curcuminoids in the treatment of inflammation associated neurodegenerative diseases.

Hypoxia enhances antibody-dependent dengue virus infection.

Dengue virus (DENV) has been found to replicate in lymphoid organs such as the lymph nodes, spleen, and liver in post-mortem analysis. These organs are known to have low oxygen levels (~0.5-4.5% O2) due to the vascular anatomy. However, how physiologically low levels of oxygen affect DENV infection via hypoxia-induced changes in the immune response remains unknown. Here, we show that monocytes adapted to 3% O2 show greater susceptibility to antibody-dependent enhancement of DENV infection. Low oxygen level induces HIF1α-dependent upregulation of fragment crystallizable gamma receptor IIA (FcγRIIA) as well as HIF1α-independent alterations in membrane ether lipid concentrations. The increased FcγRIIA expression operates synergistically with altered membrane composition, possibly through increase membrane fluidity, to increase uptake of DENV immune complexes for enhanced infection. Our findings thus indicate that the increased viral burden associated with secondary DENV infection is antibody-dependent but hypoxia-induced and suggest a role for targeting hypoxia-induced factors for anti-dengue therapy.

Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs.

Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.

Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

Comprehensive analysis of lipid composition in crude palm oil using multiple lipidomic approaches.

Palm oil is currently the leading edible oil consumed worldwide. Triacylglycerol (TAG) and diacylglycerol (DAG) are the dominant lipid classes in palm oil. Other lipid classes present in crude palm oil, such as phospholipids and galactolipids, are very low in abundance. These low-abundance lipids constitute key intermediates in lipid biosynthesis. In this study, we applied multiple lipidomic approaches, including high-sensitivity and high-specificity multiple reaction monitoring, to comprehensively quantify individual lipid species in crude palm oil. We also established a new liquid chromatography-coupled mass spectrometry method that allows direct quantification of low-abundance galactolipids in palm oil without the need for sample pretreatment. As crude palm oil contains large amounts of neutral lipids, our direct-detection method circumvents many of the challenges encountered with conventional lipid quantification methods. This approach allows direct measurement of lipids with no hassle during sample preparation and is more accurate and precise compared with other methods.

Transcriptomic and lipidomic profiles of glycerolipids during Arabidopsis flower development.

Flower glycerolipids are the yet-to-be discovered frontier of the lipidome. Although ample evidence suggests important roles for glycerolipids in flower development, stage-specific lipid profiling in tiny Arabidopsis flowers is challenging. Here, we utilized a transgenic system to synchronize flower development in Arabidopsis. The transgenic plant PAP1::AP1-GR ap1-1 cal-5 showed synchronized flower development upon dexamethasone treatment, which enabled massive harvesting of floral samples of homogenous developmental stages for glycerolipid profiling. Glycerolipid profiling revealed a decrease in concentrations of phospholipids involved in signaling during the early development stages, such as phosphatidic acid and phosphatidylinositol, and a marked increase in concentrations of nonphosphorous galactolipids during the late stage. Moreover, in the midstage, phosphatidylinositol 4,5-bisphosphate concentration was increased transiently, which suggests the stimulation of the phosphoinositide metabolism. Accompanying transcriptomic profiling of relevant glycerolipid metabolic genes revealed simultaneous induction of multiple phosphoinositide biosynthetic genes associated with the increased phosphatidylinositol 4,5-bisphosphate concentration, with a high degree of differential expression patterns for genes encoding other glycerolipid-metabolic genes. The phosphatidic acid phosphatase mutant pah1 pah2 showed flower developmental defect, suggesting a role for phosphatidic acid in flower development. Our concurrent profiling of glycerolipids and relevant metabolic gene expression revealed distinct metabolic pathways stimulated at different stages of flower development in Arabidopsis.

The mitochondria-targeted antioxidant MitoQ extends lifespan and improves healthspan of a transgenic Caenorhabditis elegans model of Alzheimer disease.

ß-Amyloid (Aß)-induced toxicity and oxidative stress have been postulated to play critical roles in the pathogenic mechanism of Alzheimer disease (AD). We investigated the in vivo ability of a mitochondria-targeted antioxidant, MitoQ, to protect against Aß-induced toxicity and oxidative stress in a Caenorhabditis elegans model overexpressing human Aß. Impairment of electron transport chain (ETC) enzymatic activity and mitochondrial dysfunction are early features of AD. We show that MitoQ extends lifespan, delays Aß-induced paralysis, ameliorates depletion of the mitochondrial lipid cardiolipin, and protects complexes IV and I of the ETC. Despite its protective effects on lifespan, healthspan, and ETC function, we find that MitoQ does not reduce DCFDA fluorescence, protein carbonyl levels or modulate steadystate ATP levels or oxygen consumption rate. Moreover, MitoQ does not attenuate mitochondrial DNA (mtDNA) oxidative damage. In agreement with its design, the protective effects of MitoQ appear to be targeted specifically to the mitochondrial membrane and our findings suggest that MitoQ may have therapeutic potential for Aß- and oxidative stress-associated neurodegenerative disorders, particularly AD.

Specific inhibition of p25/Cdk5 activity by the Cdk5 inhibitory peptide reduces neurodegeneration in vivo.

The aberrant hyperactivation of Cyclin-dependent kinase 5 (Cdk5), by the production of its truncated activator p25, results in the formation of hyperphosphorylated tau, neuroinflammation, amyloid deposition, and neuronal death in vitro and in vivo. Mechanistically, this occurs as a result of a neurotoxic insult that invokes the intracellular elevation of calcium to activate calpain, which cleaves the Cdk5 activator p35 into p25. It has been shown previously that the p25 transgenic mouse as a model to investigate the mechanistic implications of p25 production in the brain, which recapitulates deregulated Cdk5-mediated neuropathological changes, such as hyperphosphorylated tau and neuronal death. To date, strategies to inhibit Cdk5 activity have not been successful in targeting selectively aberrant activity without affecting normal Cdk5 activity. Here we show that the selective inhibition of p25/Cdk5 hyperactivation in vivo, through overexpression of the Cdk5 inhibitory peptide (CIP), rescues against the neurodegenerative pathologies caused by p25/Cdk5 hyperactivation without affecting normal neurodevelopment afforded by normal p35/Cdk5 activity. Tau and amyloid pathologies as well as neuroinflammation are significantly reduced in the CIP-p25 tetra transgenic mice, whereas brain atrophy and subsequent cognitive decline are reversed in these mice. The findings reported here represent an important breakthrough in elucidating approaches to selectively inhibit the p25/Cdk5 hyperactivation as a potential therapeutic target to reduce neurodegeneration.

Chondroitin fragments are odorants that trigger fear behavior in fish.

The ability to detect and avoid predators is essential to survival. Various animals, from sea urchins to damselfly larvae, use injury of conspecifics to infer the presence of predators. In many fish, skin damage causes the release of chemicals that elicit escape and fear in members of the shoal. The chemical nature of the alarm substance ("Schreckstoff" in German), the neural circuits mediating the complex response, and the evolutionary origins of a signal with little obvious benefit to the sender, are unresolved. To address these questions, we use biochemical fractionation to molecularly characterize Schreckstoff. Although hypoxanthine-3 N-oxide has been proposed to be the alarm substance, it has not been reliably detected in the skin and there may be other active components. We show that the alarm substance is a mixture that includes the glycosaminoglycan (GAG) chondroitin. Purified chondroitins trigger fear responses. Like skin extract, chondroitins activate the mediodorsal posterior olfactory bulb, a region innervated by crypt neurons that has a unique projection to the habenula. These findings establish GAGs as a new class of odorants in fish, which trigger alarm behavior possibly via a specialized circuit.

Cdk5/p25-induced cytosolic PLA2-mediated lysophosphatidylcholine production regulates neuroinflammation and triggers neurodegeneration.

The deregulation of cyclin-dependent kinase 5 (Cdk5) by p25 has been shown to contribute to the pathogenesis in a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). In particular, p25/Cdk5 has been shown to produce hyperphosphorylated tau, neurofibrillary tangles as well as aberrant amyloid precursor protein processing found in AD. Neuroinflammation has been observed alongside the pathogenic process in these neurodegenerative diseases, however the precise mechanism behind the induction of neuroinflammation and the significance in the AD pathogenesis has not been fully elucidated. In this report, we uncover a novel pathway for p25-induced neuroinflammation where p25 expression induces an early trigger of neuroinflammation in vivo in mice. Lipidomic mass spectrometry, in vitro coculture and conditioned media transfer experiments show that the soluble lipid mediator lysophosphatidylcholine (LPC) is released by p25 overexpressing neurons to initiate astrogliosis, neuroinflammation and subsequent neurodegeneration. Reverse transcriptase PCR and gene silencing experiments show that cytosolic phospholipase 2 (cPLA2) is the key enzyme mediating the p25-induced LPC production and cPLA2 upregulation is critical in triggering the p25-mediated inflammatory and neurodegenerative process. Together, our findings delineate a potential therapeutic target for the reduction of neuroinflammation in neurodegenerative diseases including AD.

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28.

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 µg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 µg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 µg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 µg/ml to 15 µg/ml with a GI(50) of 0.58 µg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 µg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.

Derivatization-independent cholesterol analysis in crude lipid extracts by liquid chromatography/mass spectrometry: applications to a rabbit model for atherosclerosis.

Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes.

Mitochondrial changes in ageing Caenorhabditis elegans--what do we learn from superoxide dismutase knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the "vicious cycle" theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the "vicious cycle" theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the "vicious cycle" theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.

Comparative plasma lipidome between human and cynomolgus monkey: are plasma polar lipids good biomarkers for diabetic monkeys?

BACKGROUND: Non-human primates (NHP) are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis. METHODOLOGIES/PRINCIPAL FINDINGS: We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS). We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3). Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005) and plasmalogen PC species (p<0.0005), while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA) in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005) and GM(3) (p<0.0005), but higher level of Cer (p<0.0005) in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls. CONCLUSIONS: For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys exhibited decreased levels of sphingolipids, which are microdomain-associated lipids and are thought to be associated with insulin sensitivity. Significant increases in PG species, which are precursors for cardiolipin biosynthesis in mitochondria, were found in fasted diabetic monkeys (n = 8).

Lipid pathway alterations in Parkinson's disease primary visual cortex.

BACKGROUND: We present a lipidomics analysis of human Parkinson's disease tissues. We have focused on the primary visual cortex, a region that is devoid of pathological changes and Lewy bodies; and two additional regions, the amygdala and anterior cingulate cortex which contain Lewy bodies at different disease stages but do not have as severe degeneration as the substantia nigra. METHODOLOGY/PRINCIPAL FINDINGS: Using liquid chromatography mass spectrometry lipidomics techniques for an initial screen of 200 lipid species, significant changes in 79 sphingolipid, glycerophospholipid and cholesterol species were detected in the visual cortex of Parkinson's disease patients (n = 10) compared to controls (n = 10) as assessed by two-sided unpaired t-test (p-value <0.05). False discovery rate analysis confirmed that 73 of these 79 lipid species were significantly changed in the visual cortex (q-value <0.05). By contrast, changes in 17 and 12 lipid species were identified in the Parkinson's disease amygdala and anterior cingulate cortex, respectively, compared to controls; none of which remained significant after false discovery rate analysis. Using gas chromatography mass spectrometry techniques, 6 out of 7 oxysterols analysed from both non-enzymatic and enzymatic pathways were also selectively increased in the Parkinson's disease visual cortex. Many of these changes in visual cortex lipids were correlated with relevant changes in the expression of genes involved in lipid metabolism and an oxidative stress response as determined by quantitative polymerase chain reaction techniques. CONCLUSIONS/SIGNIFICANCE: The data indicate that changes in lipid metabolism occur in the Parkinson's disease visual cortex in the absence of obvious pathology. This suggests that normalization of lipid metabolism and/or oxidative stress status in the visual cortex may represent a novel route for treatment of non-motor symptoms, such as visual hallucinations, that are experienced by a majority of Parkinson's disease patients.