ip

Parenteral lipids shape gut bile acid pools and microbiota profiles in the prevention of cholestasis in preterm pigs

Lee Call
Apr 29, 2020; 0:jlr.RA120000652v1-jlr.RA120000652
Research Articles




ip

Sphingolipids distribution at mitochondria-associated membranes (MAM) upon induction of apoptosis.

Vincent Mignard
Apr 29, 2020; 0:jlr.RA120000628v1-jlr.RA120000628
Research Articles




ip

A Direct Fluorometric Activity Assay for Lipid Kinases and Phosphatases

Jiachen Sun
Apr 27, 2020; 0:jlr.D120000794v1-jlr.D120000794
Methods




ip

Heritability of 596 lipid species and genetic correlation with cardiovascular traits in the Busselton Family Heart Study

Gemma Cadby
Apr 1, 2020; 61:537-545
Patient-Oriented and Epidemiological Research




ip

Phosphatidylinositol Metabolism, Phospholipases, Lipidomics, and Cancer:In Memoriam of Michael J. O. Wakelam (1955-2020)

Edward A Dennis
Apr 28, 2020; 0:jlr.T120000868v1-jlr.T120000868
Tribute




ip

Dynamics of sphingolipids and the serine palmitoyltransferase complex in rat oligodendrocytes during myelination

Deanna L. Davis
Apr 1, 2020; 61:505-522
Research Articles




ip

A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound

Timothy D Rohrbach
Apr 27, 2020; 0:jlr.D120000809v1-jlr.D120000809
Methods




ip

ANGPTL4 inactivates lipoprotein lipase by catalyzing the irreversible unfolding of LPLs hydrolase domain

Kristian K Kristensen
Apr 23, 2020; 0:jlr.ILR120000780v1-jlr.ILR120000780
Images in Lipid Research




ip

The grease trap: uncovering the mechanism of the hydrophobic lid in Cutibacterium acnes lipase

Hyo Jung Kim
May 1, 2020; 61:722-733
Research Articles




ip

A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

Shwetha K. Shetty
Apr 1, 2020; 61:546-559
Methods




ip

Roles of endogenous ether lipids and associated PUFA in the regulation of ion channels and their relevance for disease

Delphine Fontaine
Apr 7, 2020; 0:jlr.RA120000634v1-jlr.RA120000634
Research Articles




ip

The lncRNA Gm15622 stimulates SREBP-1c expression and hepatic lipid accumulation by sponging the miR-742-3p in mice

Minjuan Ma
Mar 30, 2020; 0:jlr.RA120000664v1-jlr.RA120000664
Research Articles




ip

LDL subclass lipidomics in atherogenic dyslipidemia:Effect of statin therapy on bioactive lipids and dense LDL

M John Chapman
Apr 15, 2020; 0:jlr.P119000543v1-jlr.P119000543
Patient-Oriented and Epidemiological Research




ip

Alcohol effects on hepatic lipid metabolism

Sookyoung Jeon
Apr 1, 2020; 61:470-479
Reviews




ip

Dietary plant stanol ester supplementation reduces peripheral symptoms in a mouse model of Niemann-Pick type C1 disease.

Inês Magro dos Reis
Apr 14, 2020; 0:jlr.RA120000632v1-jlr.RA120000632
Research Articles




ip

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

Inga Nilsson
Mar 10, 2020; 0:jlr.RA120000654v1-jlr.RA120000654
Research Articles




ip

The fatty acids from LPL-mediated processing of triglyceride-rich lipoproteins are taken up rapidly by cardiomyocytes

Haibo Jiang
Apr 2, 2020; 0:jlr.ILR120000783v1-jlr.ILR120000783
Images in Lipid Research




ip

Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities

Jeroen van Smeden
Apr 7, 2020; 0:jlr.RA120000639v1-jlr.RA120000639
Research Articles




ip

Lipid rafts as a therapeutic target

Dmitri Sviridov
May 1, 2020; 61:687-695
Thematic Reviews




ip

Dispersed lipid droplets: an intermediate site for lipid transport and metabolism in primary human adipocytes.

Björn Morén
Apr 15, 2020; 0:jlr.ILR120000808v1-jlr.ILR120000808
Images in Lipid Research




ip

GPIHBP1, a partner protein for lipoprotein lipase, is expressed only in capillary endothelial cells

Xia Meng
May 1, 2020; 61:591-591
Images in Lipid Research




ip

Images in Lipid Research

Stephen G. Young
May 1, 2020; 61:589-590
Editorials




ip

Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes [Molecular Bases of Disease]

Zinc is a critical element for insulin storage in the secretory granules of pancreatic beta cells. The islet-specific zinc transporter ZnT8 mediates granular sequestration of zinc ions. A genetic variant of human ZnT8 arising from a single nonsynonymous nucleotide change contributes to increased susceptibility to type-2 diabetes (T2D), but it remains unclear how the high risk variant (Arg-325), which is also a higher frequency (>50%) allele, is correlated with zinc transport activity. Here, we compared the activity of Arg-325 with that of a low risk ZnT8 variant (Trp-325). The Arg-325 variant was found to be more active than the Trp-325 form following induced expression in HEK293 cells. We further examined the functional consequences of changing lipid conditions to mimic the impact of lipid remodeling on ZnT8 activity during insulin granule biogenesis. Purified ZnT8 variants in proteoliposomes exhibited more than 4-fold functional tunability by the anionic phospholipids, lysophosphatidylcholine and cholesterol. Over a broad range of permissive lipid compositions, the Arg-325 variant consistently exhibited accelerated zinc transport kinetics versus the Trp-form. In agreement with the human genetic finding that rare loss-of-function mutations in ZnT8 are associated with reduced T2D risk, our results suggested that the common high risk Arg-325 variant is hyperactive, and thus may be targeted for inhibition to reduce T2D risk in the general populations.




ip

Episode 51 - The Internet of Sick Burns (IoSB) Snapchat IPO, Android Wear 2.0 and Apple in India

Techworld Editor Charlotte Jee conducts the tech orchestra this week, trying to get a tune out of this cold, creaking week. Online Editor at Computerworld UK Scott Carey lets us know all about Snapchat's upcoming IPO, billions of dollars, and the gang comes to the realisation they'll perhaps never understand what it's for. Then Senior Staff Writer at PC Advisor Henry Burrell recaps on Google and LG's launch of Android Wear 2.0 and ask - frankly - is it a big deal? Finally Acting Editor at Macworld UK David Price discusses Apple's rumoured plans to move manufacturing to India, which inevitably moves us on to Tech Trumps.  


See acast.com/privacy for privacy and opt-out information.




ip

Episode 55 - The Internet of Leaking Everything (IoLE) New iPads, Vault 7 and Nintendo Switch woes

The big story of the week is Wikileaks' CIA data dump, and we sandwich that topic between Apple's upcoming event and Nintendo Switch hardware issues. Lewis Painter kicks things off with Apple's (fingers crossed) late March event where we hope to see iPads, iMacs and get our six monthly fix of Jony Ive product videos. Then (13 minutes) Tamlin Magee tackles Vault 7 and the CIA's apparent ability to take control of iPhones and TVs, bypassing encryption. Will continuous leaks change anything? Finally (26 minutes) Dom Preston talks about his time over the last few weeks playing Zelda on the Nintendo Switch and why we should be a bit concerned about dodgy controllers.  


See acast.com/privacy for privacy and opt-out information.




ip

Episode 57 - The Internet of Apple press releases (IoAPR) iPads, red iPhones, black OnePlus phones and Android O

Gather round as three men spend 30 minutes talking about tech. Wait, what do you mean 'no thanks'?! Matt Egan shares the mic with David Price and Henry Burrell to thrash out why Apple deemed a press release adequate to announce the successor to 2014's iPad Air 2. There's also a slick new red iPhone, and a black OnePlus 3T. What's with the limited editions, eh? Or are they even limited? The gang also chat about the developer preview of Android O, out now while Android N is barely months old. Is it too early to say if it'll kick ass, and why do Google and Apple need such long public lead time with new OS builds?  


See acast.com/privacy for privacy and opt-out information.




ip

Episode 60 - The Internet of post-Easter nerds (IoPEN) F8, Mac Pro and RIP NES Classic

Back with a bumper edition after the Easter break, as Henry Burrell takes Scott Carey, David Price and Dom Preston on a chat odyssey to discuss Facebook's F8 conference. Will chat bots ever be good and who uses QR codes? The gang then discusses Apple's out of character decision to brief journos on the Mac Pro and even admit they got it wrong. Finally we talk about Nintendo stopping production of the NES Classic and whether there's more affordable retro goodness around the corner.  


See acast.com/privacy for privacy and opt-out information.




ip

Episode 69 - The Internet of internal memos (IoIM) Google sexism row and iPhone 8 leaks

Join Miriam Harris, David Price and Henry Burrell as they dissect the weird internal memo row at Google. Was a male engineer genuinely trying to help Google and make a point about the company being too Left, or is he just an arsehole? Then we describe the iPhone 8 dummy we received this week (take a look at it here: https://www.youtube.com/watch?v=PRNIwh61j_E ) and the revelations that came from the Homepod firmware leak. We also debate the usefulness of an LTE Apple Watch and try to make puns work. Magic.  


See acast.com/privacy for privacy and opt-out information.




ip

Episode 71: The Internet of iPhone X (IoiX) Plus Apple Watch 3 and Apple TV announcements

This week it is all about Apple as the company announces its eagerly awaited iPhone X and iPhone 8/8 Plus smartphones, a new Apple watch (yawn) and Apple TV news. Macworld UK editor Karen Haslam is our host this week, as she chats to a distinctly unconvinced Dominic Preston about the latest iPhone, followed by David Price on the Apple Watch Series 3, which he likes but not as much as its predecessor. Then everyone piles in together to discuss the quite-good iPhone 8, the quite-late Apple TV 4K, and all the rest of the evening's festivities.  


See acast.com/privacy for privacy and opt-out information.




ip

Episode 75 - The Internet of Driverless Pods (IoDP) Hands on with the iPhone X, FairPhone and Driverless Car design

After a lengthy hiatus we are back to FINALLY talk about the iPhone X. Computerworld UK editor Scott Carey is in the hosting chair to chat with Chris Martin, reviews editor at Tech Advisor and Macworld UK, now that we have got our hands on one. Chris talks us through what he likes and dislikes so far about the pricey smartphone.


Then Miriam Harris, staff writer at Digital Arts jumps in to discuss the design principles behind driverless cars and what we can expect in the future (17:00).


Finally, pod debutante Caroline Vanier, senior staff writer at Tech Advisor France talks about the FairPhone and if it is a new model for the smart phone industry (28:00).

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 77 - The Internet of Tulips (IoT) Bitcoin, the platform wars and Christmas tech gifts

We finally get the IoT acronym the pod has been waiting for as we discuss Bitcoin and 17th century Dutch finance. Charlotte Jee informs Henry Burrell, David Price and Scott Carey.


Scott then leads us down the winding road of the so-called platform wars, in a week when Google blocked the Amazon Echo Show from streaming YouTube. Is this a bump in the road or will services be cut from rival hardware?


And finally, led by David, the pod discusses its favourite tech of the year and what you might consider buying for friends and family for Christmas.

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 79 - The Internet of New Year (IoNY) Meltdown & Spectre, iPhone batteries, iMac Pro and the VFX Bafta noms

2018 lands with a distinct thud as Charlotte Jee tackles Meltdown and Spectre, David Price wrestles with Apple's batteries and its new iMac Pro, before Miriam Harris works through the Bafta nominations for visual effects. Henry Burrell leads us down the rabbit hole.

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 86 - The Internet of Education (IoE) New iPads for schools and the Huawei P20

Sheriff Scott Carey rounds up his two deputies in David Price and Henry Burrell for a quick-fire two topic pod. Apple launched a new cheaper iPad this week based around its education play - but can schools afford them and are Chromebooks a better option?


Then we discuss the new Huawei P20 and P20 Pro. Notches, three cameras but competitive pricing make the new Android flagship an interesting option.

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 102 - The Internet of Tennis Max (IoTM) Apple event special, new iPhones and Apple Watch

A quick-fire reaction Apple special, recorded the morning after new iPhones and an Apple Watch were announced at an event in California.


Henry Burrell joins David Price, Ashleigh Macro and Scott Carey to discuss which products we'll be buying, avoiding and coveting over the Christmas period.


Is the new iPhone named terribly? Is it an upgrade at all? What wasn't announced? And does Scott give the new Apple Watch any praise at all? It's all here.

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 104 - The Internet of Circles (IoC) RIP Google+, Pixel 3 and new tech in films

This week our host Scott Carey catches up on the Google+ breach news and the final demise of the doomed social media network before being joined by consumer technology editor at Tech Advisor, Henry Burrell, to talk about Google's latest batch of smartphones: the Pixel 3 and Pixel 3 XL.


Then Techworld reporter Tamlin Magee joins to talk about the technology-related films screening during the London Film Festival this month and his hopes for more utopian tech-flecked stories in the future.

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 105 - The Internet of Good & Bad (IoG&B) Google Rubin scandal and iPhone XR hands-on

It's episode 105, and David Price takes on hosting duties as the team dissect the best and absolute worst of the week's tech developments.


Scott Carey talks us through the troubling and wide-ranging allegations of sexual misconduct at Google, and discusses how this relates to the tech industry's culture of protected privilege, and to the #metoo movement.


Then Henry Burrell, fresh from a briefing at Apple HQ, reports his distinctly positive first impressions of the iPhone XR, which he would gladly recommend to tech newbie grandmas, and also to Scott.

 

See acast.com/privacy for privacy and opt-out information.




ip

Episode 111 - The Internet of Not Watching Films (IoNWF) Bird Box, Black Mirror and bent iPads

Happy New Year from the UK Tech Weekly Podcast!


We are back this week to discuss what we have been watching (or not watching) over the festive period, including Scott Carey on the meme-marketed sensation of Bird Box and what this says about Netflix's ability to dominate the cultural conversation. Then Tamlin Magee jumps in to talk about the interactive Black Mirror movie Bandersnatch and why it may have been destined to fail.


Lastly David Price steps out of the hosting chair to talk about bendy iPads and Apple's strange rhetoric.

 

See acast.com/privacy for privacy and opt-out information.




ip

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes [Research Articles]

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPS) on the outer leaflet and phospholipids (PLs) on the inner leaflet. Loss of this asymmetry due to mutations in the lipopolysaccharide (LPS) biosynthesis or transport pathways causes externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we employed metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase (Pcs) expression in Escherichia coli allowed for incorporation of exogenous propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC) and for incorporation of exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC) as confirmed by LC-MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells, but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains, and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool to detect a compromised bacterial OM, reveal aberrant PL externalization, and identify or characterize novel cell-active inhibitors of LPS biosynthesis or transport.




ip

The lncRNA Gm15622 stimulates SREBP-1c expression and hepatic lipid accumulation by sponging the miR-742-3p in mice [Research Articles]

Excessive lipid deposition is a hallmark of nonalcoholic fatty liver disease (NAFLD). Although much has been learned about the enzymes and metabolites involved in NAFLD, few studies have focused on the role of long non-coding RNAs (lncRNAs) in hepatic lipid accumulation. Here, using in vitro and in vivo models of NAFLD, we found that the lncRNA Gm15622 is highly expressed in the liver of obese mice fed a high-fat diet (HFD) and in murine liver (AML-12) cells treated with free fatty acids. Investigating the molecular mechanism in the liver-enriched expression of Gm15622 and its effects on lipid accumulation in hepatocytes and on NAFLD pathogenesis, we found that Gm15622 acts as a sponge for the microRNA miR-742-3p. This sponging activity increased the expression of the transcriptional regulator sterol regulatory element–binding transcription factor 1c (SREBP-1c) and promoted lipid accumulation in the liver of the HFD mice and AML-12 cells. Moreover, further results indicated that metformin suppresses Gm15622 and alleviates NAFLD-associated lipid deposition in mice. In conclusion, we have identified an lncRNA Gm15622–miR-742-3p–SREBP-1c regulatory circuit associated with NAFLD in mice, a finding that significantly advances our insight into how lipid metabolism and accumulation are altered in this metabolic disorder. Our results also suggest that Gm15622 may be a potential therapeutic target for managing NAFLD.




ip

The fatty acids from LPL-mediated processing of triglyceride-rich lipoproteins are taken up rapidly by cardiomyocytes [Images in Lipid Research]




ip

Roles of endogenous ether lipids and associated PUFA in the regulation of ion channels and their relevance for disease [Research Articles]

Ether lipids (ELs) are lipids characterized by the presence of either an ether linkage (alkyl lipids) or a vinyl ether linkage (i.e. plasmalogens [Pls]) at the sn1 position of the glycerol backbone and they are enriched in PUFAs at the sn2 position. In this review, we highlight that ELs have various biological functions, act as a reservoir for second messengers (such as PUFAs), and have roles in many diseases. Some of the biological effects of ELs may be associated with their ability to regulate ion channels that control excitation-contraction/secretion/mobility coupling and therefore cell physiology. These channels are embedded in lipid membranes, and lipids can regulate their activities directly or indirectly as second messengers or by incorporating into membranes. Interestingly, ELs and EL-derived PUFAs have been reported to play a key role in several pathologies, including neurological disorders, cardiovascular diseases, and cancers. Investigations leading to a better understanding of their mechanisms of action in pathologies have opened a new field in cancer research. In summary, newly identified lipid regulators of ion channels, such as ELs and PUFAs, may represent valuable targets to improve disease diagnosis and advance the development of new therapeutic strategies for managing a range of diseases and conditions.




ip

Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities [Research Articles]

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) stratum corneum ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active, epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in stratum corneum ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered stratum corneum ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. 




ip

Dietary plant stanol ester supplementation reduces peripheral symptoms in a mouse model of Niemann-Pick type C1 disease. [Research Articles]

Niemann–Pick type C1 (NPC1) disease is a rare genetic condition in which the function of the lysosomal cholesterol transporter NPC1 protein is impaired. Consequently, sphingolipids and cholesterol accumulate in lysosomes of all tissues, triggering a cascade of pathological events that culminate in severe systemic and neurological symptoms. Lysosomal cholesterol accumulation is also a key-factor in the development of atherosclerosis and non-alcoholic steatohepatitis (NASH). In these two metabolic diseases, the administration of plant stanol esters has been shown to ameliorate cellular cholesterol accumulation and inflammation. Given the overlap of pathological mechanisms among atherosclerosis, NASH and NPC1 disease, we sought to investigate whether dietary supplementation with plant stanol esters improves the peripheral features of NPC1 disease. To this end, we used an NPC1 murine model featuring an Npc1 null allele (Npc1nih), creating a dysfunctional NPC1 protein. Npc1nih mice were fed a two or six percent plant stanol esters–enriched diet over the course of 5 weeks. During this period, hepatic and blood lipid and inflammatory profiles were assessed. Npc1nih mice fed the plant stanol–enriched diet exhibited lower hepatic cholesterol accumulation, damage and inflammation than regular chow–fed Npc1nih mice. Moreover, plant stanol consumption shifted circulating T-cells and monocytes in particular towards an anti-inflammatory profile. Overall, these effects were stronger following dietary supplementation with 6% stanols, suggesting a dose-dependent effect. The findings of our study highlight the potential use of plant stanols as an affordable complementary means to ameliorate disorders in hepatic and blood lipid metabolism and reduce inflammation in NPC1 disease.




ip

LDL subclass lipidomics in atherogenic dyslipidemia:Effect of statin therapy on bioactive lipids and dense LDL [Patient-Oriented and Epidemiological Research]

Atherogenic LDL particles are physicochemically and metabolically heterogeneous. Can bioactive lipid cargo differentiate LDL subclasses, and thus potential atherogenicity?  What is the effect of statin treatment? Obese, hypertriglyceridemic, hypercholesterolemic males (n=12; Lp(a) <10 mg/dL) received pitavastatin calcium (4mg/day) for 180 days in a single-phase, unblinded study. The lipidomic profiles (23 lipid classes) of five LDL subclasses fractionated from baseline and post-statin plasmas were determined by LC-MS. At baseline and on statin treatment, very small dense LDL (LDL5) was preferentially enriched (up to 3-fold) in specific lysophospholipids (lysophosphatidylcholine (LPC); lysophosphatidylinositol (LPI); lyso-platelet activating factor (LPC(O)); 9,0.2 and 0.14 mol/mol apoB respectively; all p<0.001 versus LDL1-4), suggesting  elevated inflammatory potential per particle. In contrast, lysophosphatidylethanolamine was uniformly distributed among LDL subclasses. Statin treatment markedly reduced absolute plasma concentrations of all LDL subclasses (up to 33.5%), including LPC, LPI and LPC(O) contents (up to -52%), consistent with reduction in cardiovascular risk. Despite such reductions, lipotoxic ceramide load per particle in LDL1-5 (1.5 - 3 mol/mol apoB; 3 - 7 mmol/mol phosphatidylcholine) was either conserved or elevated. Bioactive lipids may constitute biomarkers for the cardiometabolic risk associated with specific LDL subclasses in atherogenic dyslipidemia at baseline, and with residual risk on statin therapy.




ip

Dispersed lipid droplets: an intermediate site for lipid transport and metabolism in primary human adipocytes. [Images in Lipid Research]




ip

Ebola virus matrix protein VP40 hijacks the host plasma membrane to form the virus envelope [Images in Lipid Research]




ip

ANGPTL4 inactivates lipoprotein lipase by catalyzing the irreversible unfolding of LPLs hydrolase domain [Images in Lipid Research]




ip

Accessibility of cholesterol at cell surfaces [Images in Lipid Research]




ip

A Direct Fluorometric Activity Assay for Lipid Kinases and Phosphatases [Methods]

Lipid kinases and phosphatases play key roles in cell signaling and regulation, and are implicated in many human diseases, and are hence thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Due Because of to its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including Class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided the newdetailed mechanistic information about the product inhibition and substrate acyl acyl-chain selectivity of PI3K and allowed enabled rapid evaluation of its small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological functionThe assay also quantitatively determined the substrate specificity of PTEN, thereby providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.




ip

A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound [Methods]

Mass spectrometry (MS) assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of optimal cutting temperature compound (OCT) used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT also interferes with protein quantification assays, and that the presence of OCT impacts the quantification of extracted sphingolipids by LC–ESI–MS/MS. We developed and validated a simple and inexpensive method that removes OCT from OCT-embedded tissues. Our results indicate that removal of OCT from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC–ESI–MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer, and to determine the long-term stability of sphingolipids in OCT-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT-cryopreserved normal lung tissues. This validated sphingolipidomic OCT-removal protocol should be a valuable addition to the lipid biologist’s toolbox.