#function of glycerophospholipids

[leedsomics]

Multi-omics data reveals the important role of glycerophospholipid metabolism in the crosstalk between gut and brain in depression

CONCLUSION: Our results provided novel perceptions on the role of Gut-Liver-Brain axis in the onset of depression, and showed that GP metabolism might be the bridge between gut microbiota and depression. "Lachnospiraceae-GP metabolism-GABA" held the promise as a potential way between gut microbiota and brain functions in DM. http://dlvr.it/SjCTry

[rnomics]

Medicina, Vol. 59, Pages 190: Sevoflurane Exposure of Clinical Doses in Pregnant Rats Induces Vcan Changes without Significant Neural Apoptosis in the Offspring

Background and Objectives: Sevoflurane is a commonly used inhalational anaesthetic in clinics. Prolonged exposure to sevoflurane can induce significant changes in lipid metabolism and neuronal damage in the developing brain. However, the effect of exposure of pregnant rats to clinical doses of sevoflurane remains unclear. Materials and Methods: Twenty-eight pregnant rats were randomly and equally divided into sevoflurane exposure(S) group, control (C) and a blank group at gestational day (G)18; Rats in S group received 2% sevoflurane with 98% oxygen for 6 h in an anesthetizing chamber,while C group received 100% oxygen at an identical flow rate for 6 h in an identical chamber. Partial least squares discriminant analysis (PLS-DA), ultra performance liquid chromatography/time-of-flight mass spectrometry(UPLC/TOF-MS) and MetaboAnalyst were used to analysis acquire metabolomics profiles, and immunohistochemical changes of neuronalapoptosis in hippocampus and cortex of neonatal rats were also analyzed. Results: This study aimed to explore lipidomics and transcriptomics changes related to 2% sevoflurane exposure for 6 h in the developing brains of newborn offspring rats. Ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF–MS) and #RNA sequencing (#RNA-seq) analyses were used to acquire metabolomics and transcriptomics profiles. We used #RNA-seq to analyse the expression of the coding and non-coding transcripts in neural cells of the cerebral cortex. No significant differences in arterial oxygen tension (PaO2), arterial carbon dioxide tension (PaCO2), or arterial blood gas were found between the groups. The relative standard deviation (RSD) of retention times was <1.53%, and the RSDs of peak areas ranged from 2.13% to 8.51%. Base peak chromatogram (BPC) profiles showed no differences between the groups. We evaluated the partial least square-discriminant analysis (PLS-DA) model. In negative ion mode, R2X was over 70%, R2Y was over 93%, and Q2 (cum) was over 80%. Cell apoptosis was not remarkably enhanced by TUNEL and haematoxylin and eosin (HE) staining in the sevoflurane-exposed group compared to the control group (p > 0.05). Glycerophospholipid (GP) and sphingolipid metabolism disturbances might adversely influence neurodevelopment in offspring. The expression of #mRNAs (Vcan gene, related to neuronal development, function and repair) of the sevoflurane group was significantly increased in the differential genes by qRT-PCR verification. Conclusions: GP and sphingolipid metabolism homeostasis may be potential therapeutic approaches against inhalational anaesthetic-induced neurodegenerative disorders. Meanwhile, sevoflurane-induced Vcan changes indicated some lipidomic and transcriptomic changes, even if neural cell apoptosis was not significantly changed in the usual clinical dose of sevoflurane exposure. https://www.mdpi.com/1648-9144/59/2/190?utm_source=dlvr.it&utm_medium=tumblr

[lifesciencepotluck]

Evil enzymes and biochemical bombs

Now that I have covered the ripper toxins and neurotoxins, it is time to take about enzymes. Ah, enzymes. They help digest food, catalyze important reactions, destroy pathetic prey—yes, anemones and other venomous animals have somehow managed to use enzymes to kill things. In anemones, there are two major kinds of enzymes which are classified as toxins: phospholipases A2 (PLA2s) and metalloproteases. Aside from anemones, metalloproteases are also found in as snakes and ticks. These diabolical toxins cause hemorrhage and necrosis by destroying the extracellular matrix (ECM). The ECM is a vital piece of biological architecture which provides structural and biochemical support for cells. It is composed of collagen, enzymes, and glycoproteins. All of these lovely proteins get degraded by the anemone’s merciless metalloproteases.

 Not only do anemone wreck cells with ripper toxins and use neurotoxins to attack ion channels, but they also destroy the support structure for cells. Why not attack cell membranes and myelin with phospholipases A2 (PLA2s)?

PLA2 toxins catalyze the hydrolysis of 2-acyl ester bonds glycerophospholipids. Yes, even the cell membranes aren’t safe from an anemone’s venom. The funny thing is that your pancreas uses PLA2 to digest that sandwich you had for breakfast, but a lot of venomous animals use PLA2 to kill things. PLA2 toxins could serve a bunch of different roles like defense, immobilization, and digestion. The literature suggests that PLA2 could play multiple roles which makes it a bit to tricky study the structure-function relationship of these toxins.

Figure 1: Predicted strcuture of UcPLA2. The toxin was isolated from the starlet sea anemone (Nematostella vectensis) [Source: Razpotnik, A. et al, 2010]

Just like potassium channel toxins are classified based on structure, PLA2 toxins are also classified based on structural features into four categories: phospholipase A1, phospholipase B, phospholipase C, and phospholipase D. There is a secreted family of PLA2 which need calcium ions to be active. There are certain types of PLA2’s which are found in the cytosol. They can depend on calcium ions for activity, or they can be strong independent enzymes.

Aside from a dash of potent peptides anemone venom has other intresting ingredients. 

You see, almost every component in the anemone’s venom plays a part in making sure that the prey or predator dies a slow and excruciatingly painful death. In some cases, anemone venom can also have a pinch of 5-hydroxytryptamine (5-HT, serotonin), histamine, bunodosine, and caissarone. 5-HT enhances vasodilation which enhances the impact of all the other toxins and histamine is excellent for causing sharp pain within victims. Bunodosine is an N-acylamino acid which has analgesic activity, and it targets serotonin receptors. Caissarone is a purine derivative which acts as an antagonist against adenosine receptors. The antagonistic adventures of this lovely ligand involve blocking or dampening the activity of adenosine receptors. You know what else acts as an antagonist against adenosine receptors?

Caffeine.

Figure 2:  Bunodosoma cangicum (left). Chemical structure of bunodosine (right).  [Soruce: Zaharenko, A. J. et al, 2011]

Most of my blog posts in Anemone toxins 101 focus on the toxins, but there are other exciting ligands in anemone venom which aren’t peptides. Bunodosine and caissarone could be developed into some pretty potent marine drugs. Even though I am fascinated with the biomedical applications of anemone toxins specifically, I think other components of the venom shouldn't be glossed over.

Next time, I will talk about the—New Kids on the Block.

Citations

Jouiaei, M., Yanagihara, A. A., Madio, B., Nevalainen, T. J., Alewood, P. F., & Fry, B. G. (2015). Ancient venom systems: A review on cnidaria toxins. Toxins, 7(6), 2251–2271. http://doi.org/10.3390/toxins7062251

Lehtonen, J. Y., & Kinnunen, P. K. (1995). Phospholipase A2 as a mechanosensor. Biophysical journal, 68(5), 1888-94.

Razpotnik, A. et al. A new phospholipase A2 isolated from the sea anemone Urticina crassicornis - Its primary structure and phylogenetic classification. FEBS J. 277, 2641–2653 (2010).

Zaharenko, A. J. et al. Bunodosine 391: An Analgesic Acylamino Acid from the Venom of the Sea Anemone Bunodosoma cangicum. J. Nat. Prod. 74, 378–382 (2011).

[your-dietician]

Phospholipase A2 inhibitor–loaded micellar nanoparticles attenuate inflammation and mitigate osteoarthritis progression

New Post has been published on https://depression-md.com/phospholipase-a2-inhibitor-loaded-micellar-nanoparticles-attenuate-inflammation-and-mitigate-osteoarthritis-progression/

Phospholipase A2 inhibitor–loaded micellar nanoparticles attenuate inflammation and mitigate osteoarthritis progression

INTRODUCTION

Osteoarthritis (OA) is a painful and debilitating disease of the articular cartilage, leading to joint pain, loss of joint function, and deleterious effects on the quality of daily life. It occurs in approximately 20 to 30 million adults in the United States alone, with staggering societal and economic costs (60 billion per year) (1). Current treatments for OA include nonpharmacological treatment (e.g., diet or exercise), pharmacological approaches, and surgical intervention (2). Although many pharmacologic approaches have been extensively pursued and some drugs have shown promise in preclinical studies, none has emerged with any notable clinical success, and there are no disease-modifying therapies available to delay and/or limit OA development and progression (3). Accordingly, there is a great unmet medical need to develop new interventional platforms for greater effectiveness in OA treatment.

The etiology of OA is broad and includes various mechanical, biochemical, and genetic factors (4, 5). Recent work has indicated that chronic unresolved inflammation has a critical role in OA development and progression (6, 7). Analyses of tissues from both human patients and animal models of OA have revealed that different inflammatory mediators have been implicated in OA pathogenesis (4). However, the most widely used nonsteroidal anti-inflammatory drugs only provide the short-term management of the pain symptoms in OA. Moreover, they have substantial renal and cardiovascular toxicities and limited efficacy without repeated administration (8). Thus, the identification of new anti-inflammatory targets may provide benefits in the treatment of OA disease.

Among the potent inflammatory mediators involved in the development of OA is the family of secreted phospholipase A2 (sPLA2) enzymes. sPLA2 is a heterogeneous group of enzymes that specifically recognize and catalytically hydrolyze the sn-2 ester bond of glycerophospholipids, releasing free fatty acids such as arachidonic acid (AA) and lysophospholipids (9), which are well-known mediators of inflammation and tissue damage (10–16). sPLA2 is normally present at low levels in healthy knee joint tissues. However, under pathological conditions, sPLA2 can be induced by multiple cascades and effector molecules including inflammatory cytokines (17, 18) and free radicals (19). A high expression level and activity of sPLA2 are observed in the synovial membrane, synovial fluid, and articular cartilage of human OA patients (20–23). Hence, we postulated that inhibition of sPLA2 enzyme activity by sPLA2 inhibitor (sPLA2i) can be exploited as a promising therapeutic strategy for OA treatment. Note that several sPLA2i compounds have already been developed and used in clinical trials for treating other inflammatory diseases (24). However, few studies have examined the role of sPLA2 in OA, and no study has sought to harness the sPLA2 activity for the treatment of OA (18).

Clinical management of OA is further complicated by lack of effective drug delivery systems (25, 26). Rapid clearance of the drugs by the joint (i.e., a short half-life) and therapeutic target sites deep within the cartilage not accessible to drugs pose significant delivery challenges for many promising drugs (27, 28). Recently, nanoparticle (NP)–based targeted drug delivery has been exploited in the treatment of OA (1, 27, 29). The main benefits of these delivery systems are that they can increase the retention of drugs in the joints, thus lowering the therapeutic dose, reducing the frequency of administration, increasing therapeutic efficacy, and reducing off-target toxicity. Here, we examined whether inhibition of sPLA2 enzyme activity by sPLA2i-loaded NPs could be an effective OA therapy. Specifically, a lipid-based sPLA2i, thioetheramide-PC [molecular weight (MW), 735.1 Da], was incorporated into nanometer-sized phospholipid micelles. The joint retention, cartilage penetration, biodistribution, and toxicity of the sPLA2i-loaded micellar NPs (sPLA2i-NPs) were subsequently characterized. Using an in vitro cartilage explant model and two animal models of OA, we investigated the effectiveness of sPLA2i-NPs in inhibiting the inflammatory signals and attenuating the OA progression following direct delivery into knee joints.

RESULTS

sPLA2 amount in OA cartilage

To confirm the elevated levels of sPLA2 enzyme in OA-related tissues, we performed immunohistochemistry (IHC) to investigate a commonly studied isoform sPLA2-IIA in knees. In healthy young and adult human cartilage, the level of sPLA2-IIA was very low at the top and middle region of the articular cartilage (Fig. 1, A and B). However, its amount was markedly elevated in the OA cartilage at early and middle stages. Abundant staining was found in the cartilage matrix and chondrocytes. Mouse cartilage showed similar results (Fig. 1, C and D). While normal mouse articular cartilage displayed only a weak staining of sPLA2-IIA, the cartilage in mouse knees receiving destabilization of the medial meniscus (DMM) surgery a month earlier showed greatly enhanced staining throughout the cartilage. The marked increase in sPLA2 in human and mouse OA cartilage suggested its potential role in OA development.

Fig. 1 sPLA2 expression in human and mouse OA cartilage.

(A) Representative images of Safranin O/Fast Green staining (top) and IHC of sPLA2 (bottom) in healthy juvenile, healthy adult, early-stage, and middle-stage human cartilage tissues. Scale bars, 200 μm. (B) Quantification of sPLA2-positive chondrocytes as a proportion of total chondrocytes in healthy and OA human articular cartilage tissues (n = 5). (C) Representative images of Safranin O/Fast Green staining (top) and IHC of sPLA2 (bottom) in the tibial articular cartilage of WT mice at 1 month after sham or after DMM surgery. Scale bars, 50 μm. (D) Quantification of sPLA2-positive chondrocytes in the tibial articular cartilage of sham- or DMM-operated joints (n = 5). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test for (B) and paired two-tailed t test for (D). Data presented as means ± SEM. ***P < 0.001.

sPLA2i-NP synthesis and characterization

sPLA2i-NPs were prepared by incorporating 25 mole percent (mol %) thioetheramide-PC into phospholipid micelles with lipid composition 10 mol % 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/65 mol % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) (Fig. 2A). Because of its amphiphilic nature, lipid-based sPLA2i was easily doped into the phospholipid film during NP preparation, and the sPLA2i loading efficiency was almost 100%. Dynamic light scattering (DLS) measurements revealed that sPLA2i-NPs had a mean hydrodynamic diameter of 10 nm and a relatively narrow size distribution (Fig. 2B). sPLA2i-NPs observed by transmission electron microscopy (TEM) were approximately spherical in shape together with some worm-like structures (fig. S1). As shown in Fig. 2C, the presence of cationic DOTAP within sPLA2i-loaded NPs transitioned NPs from a negative surface charge (−8 mV) to a positive surface charge (+2 mV) in phosphate-buffered saline (PBS) solution, which could increase the retention and penetration ability of sPLA2i-NPs within the cartilage via electrostatic interactions between the NPs and the anionic glycosaminoglycans (GAGs) in the cartilage. It should also be noted that the NP surface charge is highly dependent on its surrounding medium. For example, zeta potential of DOTAP-doped sPLA2i-NPs in 10 mM Hepes buffer (pH 7.4) was −14.5 mV. The surface charge of DOTAP-doped sPLA2i-NPs in synovial fluid may be different from the one in PBS or Hepes. The stability of the sPLA2i-NPs was evaluated in PBS and bovine synovial fluid. There was no observable change in the hydrodynamic diameter of sPLA2i-NPs in PBS for at least 1 week (Fig. 2D) or in bovine synovial fluid for 5 days (fig. S2).

Fig. 2 Preparation and characterization of sPLA2i-NPs.

(A) Schematic diagram of sPLA2i-NPs in which a lipid-based sPLA2i was incorporated into nanometer-sized phospholipid NPs (DSPE-PEG2000) with cationic lipid DOTAP doped. (B) DLS measurement of the hydrodynamic diameter of sPLA2i-NPs. (C) Zeta potential of sPLA2i-NPs in the presence or absence of cationic lipid DOTAP in 0.1 M PBS (pH 7.4). (D) The stability of sPLA2i-NPs in 0.1 M PBS (pH 7.4) was accessed by monitoring the hydrodynamic diameter for up to 1 week. (E) In vitro response of sPLA2i-NPs (final sPLA2i concentration, 500 nM) to sPLA2 enzyme experiment showed a significant inhibition effect. (F) The cytotoxicity of sPLA2i-NPs was determined by measuring the cell viability of primary chondrocytes after coincubation with sPLA2i-NPs at various concentrations. In all datasets, n = 3 biologically independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. Data presented as means ± SEM.

To examine whether sPLA2i-NPs can inhibit sPLA2 enzyme, a fluorescence dequenching assay with liposomes that contain a self-quenching concentration of the fluorescent lipid was used (30). Phospholipid hydrogenated soy phosphatidylcholine (HSPC) liposomes doped with 20 mol % fluorescent lipid 1-palmitoyl-2-6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl-sn-glycero-3-phosphocholine (NBD-PC) were prepared in which the NBD fluorescence was self-quenched (fig. S3). NBD-PC is a fluorescent phospholipid with the fluorophore NBD attached to the lipid backbone using an sPLA2-responsive sn-2 acyl bond. When sPLA2 enzyme was added to the NBD-PC–doped liposome sample in the absence of sPLA2i or in the presence of Ctrl-NPs (i.e., NPs without sPLA2i), sPLA2 catalytically hydrolyzed the sn-2 acyl bond of NBD-PC, resulting in the release of NBD into the surrounding bulk solution. The released NBD led to fluorescence dequenching, resulting in a higher fluorescence intensity (Fig. 2E). When sPLA2 enzyme was added to the liposome sample in the presence of sPLA2i, including free sPLA2i and sPLA2i-NPs, the reduced sPLA2 activity led to the low release of NBD into the surrounding bulk solution, resulting in a reduction in the extent of fluorescence increase (Fig. 2E). Further studies showed that free sPLA2i or sPLA2i-NPs inhibited the sPLA2 activity in a concentration-dependent manner with half-maximal inhibitory concentration (IC50) values of 2.5 and 0.59 μM, respectively (fig. S4), indicating that sPLA2 inhibition by sPLA2i-NPs was more potent than the free sPLA2i.

The cytotoxic effects of sPLA2i-NP were examined in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Specifically, various concentrations of sPLA2i-NPs were incubated with primary mouse chondrocytes for 24 hours. The cell viability for each group was normalized to a control group with no sPLA2i-NPs. In general, sPLA2i-NPs had little effect on the viability of cells up to a sPLA2i concentration of 10 μg ml−1 (Fig. 2F), suggesting their physiological biocompatibility.

sPLA2i-NP penetration

Because mouse knee cartilage is very thin and not an appropriate model for studying cartilage penetration, bovine cartilage explants, mimicking human knee cartilage (~2 to 4 mm), were used to examine whether the sPLA2i-NPs could penetrate into deep layer of cartilage tissue. sPLA2i-NPs were incubated with bovine cartilage tissue ex vivo for different times including 2, 4, 6, and 8 days. Confocal fluorescence images of cartilage section were acquired preincubation and at various time points after incubation with sPLA2i-NPs (Fig. 3A). In the preincubation images, there was little intrinsic tissue fluorescence. At 2 days following incubation with the sPLA2i-NPs, a strong fluorescence signal was observed within the superficial zone of the cartilage. The fluorescence signal was distributed in the entire cartilage tissue (1 to 2 mm in thickness) as early as day 4 and became stronger over time, indicating that the sPLA2i-NPs indeed moved into the deep zone of the cartilage. Control experiments were performed by incubation of bovine cartilage explants with sPLA2i-NPs that were not doped with the positively charged DOTAP. In these tissues, a strong fluorescence signal was only observed within the superficial zone of the cartilage, even up to 8 days. Similarly, when bovine cartilage explants were incubated with free fluorescent dye rhodamine, very little fluorescence signal was observed in the deep zone of the cartilage. Analysis of fluorescence images revealed that DOTAP-doped sPLA2i-NPs exhibited a great improvement in cartilage penetration at all time points compared with non-DOTAP–doped sPLA2i-NPs or free rhodamine (Fig. 3B and fig. S5). In addition, the area under the curve (AUC) in the cartilage achieved by sPLA2i-NPs (DOTAP+) was much larger than that of non-DOTAP–doped sPLA2i-NPs or free rhodamine (Fig. 3C and fig. S5). These results indicate that the DOTAP-doped sPLA2i-NPs are able to penetrate into the deep zone of the articular cartilage and exhibit high cartilage accumulation.

Fig. 3 Cartilage penetration and joint retention of sPLA2i-NPs.

(A) Representative confocal microscope images of cross sections of bovine cartilage explants incubated with free rhodamine dye or rhodamine-labeled sPLA2i-NPs in the presence or absence of cationic lipid DOTAP for 0, 2, 4, 6, and 8 days. Scale bar, 200 μm. (B) Semiquantitative analysis of the fluorescence intensity of the above different formulations over the entire explant sections after 8 days of incubation (n = 3). (C) Semiquantitative analysis of the AUC based on the fluorescence intensity profiles in (B) (n = 3). (D) Representative IVIS images of healthy and OA (OA was induced surgically 8 weeks before) mouse knee joints over 28 days after single intra-articular injection of free ICG or Cy7-labeled sPLA2i-NPs. (E) Semiquantitative analysis of time-course fluorescent radiant efficiency within healthy and OA mouse knee joints over 28 days (n = 5). (F) Semiquantitative analysis of the AUC based on the fluorescence intensity profiles in (E) (n = 5). Rhod-PE, free rhodamine dye; sPLA2i-NPs (DOTAP−), sPLA2i-NPs in the absence of DOTAP; sPLA2i-NPs (DOTAP+), sPLA2i-NPs in the presence of DOTAP. AU, arbitrary units. Statistical analysis was performed using one-way ANOVA with Turkey’s post hoc test. Data presented as means ± SEM. ***P < 0.001. Photo credit: Yulong Wei, University of Pennsylvania.

To further assess the total accumulation of sPLA2i-NPs within the cartilage, we acquired fluorescence images of the bovine cartilage 24 hours following the incubation of DOTAP-doped sPLA2i-NPs, non-DOTAP–doped sPLA2i-NPs, or free rhodamine. As expected, the fluorescent intensity of DOTAP-doped sPLA2i-NPs in the cartilage tissue was significantly higher than that of non-DOTAP–doped sPLA2i-NPs or free rhodamine (fig. S6, A and B). This convincingly demonstrates the benefit of including DOTAP moieties in the sPLA2i-NPs. Because of the ability to penetrate the cartilage, the DOTAP-doped sPLA2i-NPs were chosen for subsequent studies.

sPLA2i-NP retention

In vivo fluorescence imaging was used to study the retention of Cyanine 7 (Cy7)–labeled sPLA2i-NPs in the knee joints after intra-articular injection. Although this technique has limitations due to the limited depth of penetration in tissues and the sensitivity of fluorescent probes to their surrounding environment, it is still a very practical method for providing semiquantitative information for NP joint retention and in vivo biodistribution. To show the difference in retention between NPs and small-molecule agents, we used a low-MW fluorescent dye indocyanine green (ICG; MW, 775 Da) for comparison. Their retention within the joints was evaluated in the knee under healthy and early OA conditions. Fluorescence images of mouse knee joints were acquired at various time points after sample injection (Fig. 3D). With a single intra-articular injection, the fluorescence intensity of sPLA2i-NPs in joints was significantly higher than that of free ICG at all time points, indicating that the retention time of sPLA2i-NPs in joints is much longer than that of free ICG. Analysis of fluorescence images showed that the retention of sPLA2i-NPs in joints with OA condition was even greater than that of healthy joints (Fig. 3, E and F). This finding is consistent with previous reports that NPs are retained in the OA joint much longer than in the healthy joint (31, 32), possibly due to synovial thickening (31) and altered lymphatic vessels in OA knees (33). A longer retention time of sPLA2i-NPs over small molecules within knee joints was also confirmed when rats were used (fig. S7), suggesting that long retention of sPLA2i-NPs in the joints could be achieved for different animals.

We also examined the biodistribution of sPLA2i-NPs in joint components, internal organs, and blood. At 24 hours after injection, fluorescence signals were detected on the cartilage surfaces of patellar, femoral condyles, and tibial plateau as well as on the meniscus (fig. S8, A and B). By 24 hours after injection, the accumulation of the sPLA2i-NPs was mainly observed in the liver and kidney, but no signal was detected in blood at that time, indicating that sPLA2i-NPs were nearly cleared from circulation (fig. S9, A and B). One month later, no fluorescence was observed in the liver and kidney.

sPLA2i-NPs block cartilage degeneration in OA cartilage explants

The articular cartilage explants represent an in vitro model to study OA progression in a three-dimensional (3D) environment (34). We harvested femoral head cartilage explants from 2-month-old wild-type (WT) mice and then stimulated them with OA-associated proinflammatory cytokine interleukin-1β (IL-1β) (35). The penetration of sPLA2i-NPs was studied by acquiring fluorescence images of IL-1β–stimulated cartilage explants that were incubated with rhodamine-labeled sPLA2i-NPs for 24 or 48 hours. Fluorescent signal was mainly present in the cartilage surface at 24 hours (Fig. 4A), while a strong fluorescence signal was observed in the deep calcified layer at 48 hours (Fig. 4, B and C), indicating that sPLA2i-NPs are able to penetrate into femoral head cartilage treated with IL-1β. These results were consistent with those presented above for sPLA2i-NPs penetration within bovine cartilage explants.

Fig. 4 Penetration and chondroprotective effects of sPLA2i-NPs in mouse femoral heads.

(A) Representative fluorescence images of cross sections of mouse femoral heads stimulated with recombinant mouse IL-1β and incubated with rhodamine-labeled sPLA2i-NPs for 0, 24, or 48 hours. Bottom: Magnified images of the white boxed areas. Scale bars, 100 μm. (B) Semiquantitative analysis of rhodamine-labeled sPLA2i-NPs penetration depth into IL-1β–stimulated mouse femoral heads (n = 6). (C) Quantification of the AUC based on the fluorescence intensity profiles in (B) (n = 6). (D) Representative images of Safranin O/Fast Green staining on the sections of untreated and PBS-, Ctrl-NP–, sPLA2i-, and sPLA2i-NP–treated IL-1β–stimulated mouse femoral heads. Bottom: Magnified images of the black boxed areas. Scale bars, 100 μm. (E) Safranin O (Saf O)–positive area among the above groups was quantified (n = 5). (F) The OA severity was accessed by Mankin score (n = 5). (G) The relative gene expression of sPLA2-IIA, aggrecan, Col2a1, Mmp13, and Adamts5 was examined by quantitative reverse transcription polymerase chain reaction (PCR) in the untreated and PBS-, Ctrl-NP–, and sPLA2i-NP–treated IL-1β–stimulated mouse femoral heads (n = 3). Statistical analysis was performed using paired two-tailed t test for (C) and one-way ANOVA with Turkey’s post hoc test for (E), (F), and (G). Data presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

To test the therapeutic effects of sPLA2i-NPs in vitro, we divided femoral head cartilage explants into five groups that received PBS, IL-1β and PBS, IL-1β and Ctrl-NPs, IL-1β and sPLA2i, or IL-1β and sPLA2i-NP treatments, respectively, for 8 days. As shown in Fig. 4 (D to F), IL-1β treatment led to an OA-like phenotype in the cartilage, featured by surface fibrillation and proteoglycan loss. Notably, sPLA2i-NPs and free sPLA2i, but not Ctrl-NPs, blocked cartilage degeneration induced by IL-1β, leading to comparable Safranin O content and Mankin score as the untreated group. sPLA2i-NPs, but not PBS or Ctrl-NPs, attenuated IL-1β–induced sPLA2-IIA expression in the cartilage (Fig. 4G). Furthermore, the catabolic effects of IL-1β, such as decreasing the expression of matrix proteins, Col2a1 and aggrecan, and increasing the expression of proteinases, matrix metalloproteinase 13 (Mmp13), and Adamts5, were effectively reversed by sPLA2i-NPs (Fig. 4G). Together, these data provide ex vivo evidence that sPLA2i-NPs have protective action on chondrocytes against OA-inducing insults.

sPLA2i-NPs attenuate joint destruction in a surgery-induced mouse OA model

To study the in vivo therapeutic effects of sPLA2i-NPs, we used two mouse OA models. The first is a DMM surgery–induced OA model with high reliability and reproducibility (Fig. 5A). After DMM, WT knees started to show cartilage damage, including surface fibrillation and loss of proteoglycan, at 2 months, and exhibited severe cartilage erosion up to the entire uncalcified zone at 4 months, resulting in Mankin scores of 5.5 and 8.9, at these two time points, respectively (Fig. 5, B and C, and figs. S10 and S11). Intra-articular injection of sPLA2i-NPs into DMM knees, once every two weeks, starting immediately after the surgery, greatly improved the morphology and structure of the articular cartilage, leading to an almost intact cartilage surface with no proteoglycan loss even at 4 months after surgery (Fig. 5B and figs. S10 and S11B). These therapeutic effects were not observed with free sPLA2i or Ctrl-NP treatments.

Fig. 5 Therapeutic effects of sPLA2i-NPs for attenuation of DMM-induced chronic OA.

(A) The study design of sPLA2i-NP treatment for DMM-induced OA mice. (B) Safranin O/Fast Green staining of sham- or DMM-operated knee joints with 2 or 4 months of treatment. Scale bar, 200 μm. (C) The OA severity was accessed by Mankin score at 2 months after surgery (n = 8). (D) Hematoxylin and eosin (H&E) staining of synovium tissue at 2 months after surgery. Scale bar, 100 μm. Red boxed areas indicate the synovial lining layer. (E) Synovial inflammation was evaluated (n = 8). (F) 3D color maps of femoral SBP thickness at 4 months after surgery. Color ranges from 0 μm (blue) to 320 μm (red). (G) Quantification of SBP thickness (Th.) at the medial posterior site (n = 8). (H) Osteophytes (red arrows) were revealed by 2D (top) and 3D (bottom) micro–computed tomography (micro-CT) images. (I) Osteophyte volume was quantified (n = 8). (J) von Frey assay was performed at 1, 2, 4, 8, and 12 weeks after surgery (n = 8). The data of day 0 were acquired before surgery. PWT, paw withdrawal threshold. (K) IHC staining of the tibial articular cartilage at 2 months after surgery. Scale bar, 100 μm. (L) Quantification of sPLA2-IIa–, p-P65–, and p-P100–positive chondrocytes (n = 5). Statistical analysis was performed using one-way ANOVA with Turkey’s post hoc test. Data presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 in (C), (E), (G), (I), and (L). *P < 0.05 and **P < 0.01 for sPLA2i-NPs versus PBS in (J).

The thickness of synovium is an indicator of inflammation in the knee joint (36, 37). PBS-, sPLA2i-, and Ctrl-NP–treated DMM knees exhibited a significantly thickened synovial lining with more than 10-fold increase in the synovitis score compared with sham knees. In contrast, sPLA2i-NP–treated DMM knees had only a fourfold increase (Fig. 5, D and E), suggesting that sPLA2i-NPs generate an anti-inflammatory effect in the injured knees.

We further characterized some of the late OA symptoms including subchondral bone plate (SBP) sclerosis and osteophyte formation. At 4 months after DMM surgery, the thickness of the SBP at the femoral medial site in the knee joint was significantly increased in PBS-treated (1.25-fold), sPLA2i-treated (1.31-fold), and Ctrl-NP–treated (1.29-fold) groups compared to that in sham joints (Fig. 5, F and G). However, this SBP sclerosis was not observed in the sPLA2i-NP–treated group. Furthermore, osteophytes were also frequently found in DMM knees in control-treated groups but not in the sPLA2i-NP–treated group (Fig. 5, H and I). These data clearly indicate that sPLA2i-NPs prevent OA progression into a late stage.

In WT mice, mechanical allodynia was observed in the operated hind paw after DMM surgery but not following sham surgery (38). Administration of PBS, sPLA2i, or Ctrl-NPs into DMM knees did not alter the OA pain (Fig. 5J). DMM knees receiving sPLA2i-NPs initially exhibited pain response at 1 week, probably due to the persistent postsurgical pain. At 2 weeks after surgery, sPLA2i-NP treatment greatly increased the paw withdrawal threshold (PWT) to a level close to that in sham knees and maintained that level throughout the experimental period, implying that sPLA2i-NP treatment relieves OA pain.

We further studied the mechanism of sPLA2 inhibition for treating OA. IHC showed that sPLA2i-NPs blocked the up-regulation of sPLA2-IIa after OA induction (Fig. 5, K and L). Consequently, while DMM elevated the inflammatory signaling pathway, such as phospho (p)–p65 and p-p100, sPLA2i-NP treatment greatly decreased the amounts of these inflammatory indicators in the cartilage at 2 and 4 months after DMM, thus protecting cartilage from OA insults (Fig. 5, K and L, and fig. S12). Collectively, these results suggest that the sPLA2 enzyme mediates the pathogenesis of OA, while sPLA2i-NPs inhibit OA inflammation in DMM knee joints.

To assess the toxicity of sPLA2i-NPs in mice, we evaluated the histopathology of tissues from the liver, spleen, kidney, lung, heart, and knee joint of mice 2 months after serial intra-articular injections of PBS, sPLA2i, Ctrl-NPs, or sPLA2i-NPs. We did not observe any histopathological abnormalities in any of the mouse organs and knee joints that received these injections directly (figs. S13 and S14). Furthermore, we evaluated toxic effects on blood parameters of mice following treatment and did not observe any clinical signs of toxicity (fig. S15). These results suggest that sPLA2i-NPs at the adopted dosages show no toxicity in mice.

sPLA2i-NPs block joint damage in a single load-induced mouse posttraumatic OA model

The second mouse OA model we tested is a noninvasive mechanical loading model, which is clinically relevant to posttraumatic OA (PTOA) (Fig. 6A). This injury model allows for the study of early events after impact, at a time when inflammation is thought to be particularly important (29). We applied a single loading episode, composed of 60 cycles of 6- or 9-N peak load, on the mouse tibia to induce joint translation and anterior cruciate ligament (ACL) rupture. Two weeks later, a lesion was seen alongside with proteoglycan loss in the lateral femoral articular cartilage surface (Fig. 6B). Quantification of the length of cartilage injury revealed significant damage areas in PBS-, sPLA2i-, and Ctrl-NP–treated knees (Fig. 6C and fig. S16). As expected, 9 N generated more severe damage than 6 N in all groups. In this model, chondrocyte apoptosis at the loading site contributes significantly to OA development. TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining revealed that sPLA2i-NP treatment greatly reduced the number of apoptotic chondrocytes in the cartilage following loading (Fig. 6, D and E, and fig. S17). Examining the inflammatory pathway again revealed a reduction of p-P65 and p-P100 in sPLA2i-NP–treated knee joints, but not in sPLA2i or Ctrl-NP groups (Fig. 6, F and G, and fig. S18). Similar to the above DMM model, we found that sPLA2i-NPs suppressed the amount of sPLA2-IIA enzyme in the cartilage. Synovitis scores suggested significant synovitis in PBS-, sPLA2i-, and Ctrl-NP–treated knees (Fig. 6, H and I, and fig. S19). von Frey assay clearly showed that mice in these groups experienced OA pain (Fig. 6J and fig. S20). In contrast, injections of sPLA2i-NPs into knee joints immediately and 48 hours after loading reversed these PTOA symptoms regardless of loading force. Specifically, compared to PBS-treated knees, the length of cartilage injury in sPLA2i-NP–treated knees was decreased by 67% (6 N) and 59% (9 N), synovitis score was reduced by 71% (6 N) and 56% (9 N), and pain threshold was increased to a level similar to sham knees. Hence, our data demonstrate a joint protective role of sPLA2i-NPs in load-induced OA.

Fig. 6 Therapeutic effects of sPLA2i-NPs for attenuation of load-induced PTOA.

(A) The study design of sPLA2i-NP treatment for loading-induced OA mice. (B) Safranin O/Fast Green staining of the articular cartilage in sham or loaded (6 or 9 N) knee joints with 14-day treatment. Scale bar, 100 μm. Dashed lines indicate the range of loss of staining. (C) The length of cartilage lesion in sham and 6-N loaded knee joints was quantified. (D) TUNEL staining in the tibial articular cartilage from sham and 6-N loaded knee joints. Scale bar, 100 μm. (E) Quantification of TUNEL-positive chondrocytes (n = 5). (F) IHC staining of the tibial articular cartilage from sham and 6-N loaded knee joints. Scale bar, 100 μm. (G) Quantification of sPLA2-IIa–, p-P65–, and p-P100–positive chondrocytes (n = 5). (H) H&E staining of synovium tissue. Scale bar, 100 μm. Red boxed areas indicate the synovial lining layer. (I) Synovial inflammation was evaluated (n = 8). (J) von Frey assay at 1 and 2 weeks after 6-N loading (n = 8). The data of day 0 were acquired before loading. Statistical analysis was performed using one-way ANOVA with Turkey’s post hoc test. Data presented as means ± SEM. *P < 0.05 and ***P < 0.001 in (C), (E), (G), and (I) and *P < 0.05 for sPLA2i-NPs versus PBS in (J).

DISCUSSION

Growing evidence suggests that inflammation has a critical role in the pathogenesis of OA. Classical anti-inflammatory agents have limited utility to slow the progression of OA. In this work, we show that the level of sPLA2 is significantly increased in the articular cartilage in both human and mouse OA knee tissues. Because the release of free AA from membrane phospholipids by sPLA2 is one of the major contributors to producing potent inflammatory mediators such as eicosanoids or platelet-activating factor, we therefore hypothesized that sPLA2 could act as a promising therapeutic target for OA treatment. Our strategy was to develop a sPLA2i-based approach for inhibition sPLA2 activity. Compared to classical anti-inflammatory agents, inhibiting sPLA2 enzyme activity by sPLA2i could offer the potential to block production of a more complete set of inflammatory substances through targeting upstream inflammatory pathways, leading to a safer and more effective alternative to conventional anti-inflammatory agents.

Great efforts from pharmaceutical companies and academic laboratories have recently been devoted to developing potent and selective sPLA2i for the treatment of inflammation-related diseases, with several ongoing clinical trials (39). One clinical trial used the sPLA2i LY315920 to treat patients with rheumatoid arthritis (RA) (40). There was a significant short-term improvement after several days of treatment; however, no significant longer-term effect was observed after weeks of treatment (41). This failure is largely due to the lack of sufficient inhibitor concentration in the joint (41, 42), because human synovial fluid has a rapid turnover in the joint, which could flush the administrated small sPLA2i molecules out of joint through capillaries and draining lymphatic vessels (26, 43). Our results also supported this limitation (i.e., fast clearance of sPLA2i from the joint), showing that the intra-articular injection of free sPLA2i alone did not have a protective effect on cartilage degeneration in OA animals.

Drug delivery systems using NPs are increasingly being used to improve therapeutic delivery due to the favorable pharmacokinetics, biodistribution, and solubility of NPs compared with free drugs. Currently, several NPs have been developed and tested in preclinical OA studies, including liposomes, dendrimers, and poly(lactic-co-glycolic acid) particles. Because of the high density of negatively charged GAG in the cartilage, cationic drug carriers are often explored for enhancing cartilage penetration and retention based on electrostatic interactions (44–46). In this study, we have addressed the sPLA2i delivery challenges by incorporating small sPLA2i into nanometer-sized phospholipid micelles. By tuning the surface charge, the sPLA2i-NPs showed deep cartilage penetration and prolonged residence time in knee joints. This is especially important because the therapeutic target sPLA2 is located within deep cartilage tissue.

Most NPs developed and tested in preclinical OA studies, so far, have some weaknesses. Because of their relatively large size (100 to 200 nm in diameter), liposomes have prolonged residence times in joints but are inaccessible to deep cartilage tissues (47), which could become problematic if therapeutic targets (i.e., sPLA2, the therapeutic target in this study) are located within deep cartilage tissue. Therefore, liposomes are only particularly well suited for OA drug delivery to the cartilage surface (47, 48). Because of its small size and controllable surface charge, polyamidoamine (PAMAM) dendrimers can penetrate the cartilage efficiently (1). For example, Geiger et al. (1) conjugated insulin-like growth factor 1 to a cationic dendrimer, which could penetrate full-thickness bovine cartilage ex vivo. Unfortunately, thioetheramide-PC does not contain any reactive chemical groups for dendrimer conjugation. In addition, dendrimers have not translated into the clinic despite 40 years of research (49). Compared to the above NPs, phospholipid micelles we used here for loading sPLA2i have several advantages. First, sPLA2i-NPs consist almost solely of clinically used phospholipids and, thus, are readily translatable to clinical applications. Second, lipid-based sPLA2is can be incorporated into phospholipid micelles with high yield and without any conjugation or purification steps. Third, lipid-based sPLA2i-NPs are sPLA2 responsive. Fourth, the method for producing sPLA2i-NPs is highly reproducible, simple, and cost-effective because all components are included in starting materials before sPLA2i-NP formation, which would allow large-scale, good manufacturing practice (GMP) production of NPs, a necessary step for the initiation of future clinical trials. Therefore, the use of small size phospholipid micelles for loading lipid-based thioetheramide-PC is an appropriate vehicle/drug combination in the current specific settings because phospholipid micelles have a long history in clinical applications.

Inflammation plays a critical role in OA progression. Compared to rheumatoid RA, the prototypical inflammatory arthritis, the inflammation in OA is chronic and relatively low grade (50). Systemically blocking the activity of conventional inflammatory cytokines with biologic therapies approved for the treatment of RA, such as anti–tumor necrosis factor or anti–IL-1β therapies, provides no or minor benefit in generalized OA (50). These observations implicate that a new target or approach must be adopted for treating OA inflammation and resultant cartilage degeneration. Inflammation starts with the PLA2-induced hydrolysis of the membrane phospholipids, giving rise to the fatty acids, such as AA and lysophospholipids. Of all PLA2 types, sPLA2 is considered the most “inflammatory enzyme.” Past studies found that sPLA2 amount increases in human OA joints, including cartilage chondrocytes (20–23), but they did not further analyze the cause-effect relationship between sPLA2 up-regulation and OA progression. Our studies first confirmed the enhanced sPLA2 activity in both human and mouse OA cartilage. Then, we used an ex vivo bovine cartilage explant model to demonstrate that inhibiting sPLA2 protects chondrocytes against IL-1β insult via suppressing inflammation pathways. We used two in vivo mouse models of OA, which mimic different OA subtypes, to provide proof-of-principle evidence that intra-articular injections of sPLA2i-NPs are effective in attenuated OA progression. Notably, local delivery of sPLA2i-NPs, but not free sPLA2i or NP alone, greatly slowed the progression of cartilage degeneration, reduced synovial inflammation, prevented osteophyte formation, and relieved join pain. In addition to the articular cartilage, sPLA2i-NP could target other joint tissues, such as synovium and meniscus, to achieve its therapeutic effects. No signs of toxicity have been observed in the joints and other tissues, suggesting the safety of this nanomedicine. That to be said, because OA is a chronic disease, long-term treatment is required in the future to further prove its safety.

The development of sPLA2i-NP–based therapies for large animals or their translation into clinical settings requires further optimization and investigation. For example, in this work, we used thioetheramide-PC as sPLA2i due to its commercial availability and its ease of loading into phospholipid micelles. However, thioetheramide-PC is for research use only and has a relatively high IC50 (~2.5 μM). In the future, we will choose sPLA2is that have already been used in clinical trials and have a low IC50 value. There are some other variables that can be optimized to further improve the therapeutic efficacy of sPLA2i-NPs, including particle size, surface charge, hydrophobicity, and material compositions. A previous work has shown that weak-reversible binding between drug carriers and cartilage is key to allowing deep cartilage penetration (51, 52). If net surface charge of drug carriers is too high, then their diffusion rate could be slowed because of stronger binding interactions within the cartilage (53). In this work, zeta potential measurement in PBS showed a slight positive surface charge with 10 mol % DOTAP-doped sPLA2i-NPs. As expected, the DOTAP-doped sPLA2i-NPs penetrated into the deep zone of the articular cartilage. This is most likely due to a combination of the following features of NPs: favorable interactions with a slightly positive surface charge, some NPs with worm-like structures, and the modified spatial distribution of surface charge. It should be mentioned that the sPLA2i-NPs administration was initiated immediately after DMM surgery in this study. Future animal studies should incorporate injections at time points when early- or middle-stage OA symptoms appear. Given that sPLA2 level was markedly elevated in human knee cartilage at early, middle, and late stage of OA progression, it is reasonable to expect that the sPLA2i-NPs could also be used to slow cartilage degeneration and OA progression in cases where OA is already established. While our current engineered sPLA2i-NPs showed the therapeutic effects in a mouse model of OA, they may not be translated to large animals and humans with equal efficacy because the anatomy and physiology of small animals are significantly different from large animals and humans. Further optimization of this NP-based drug delivery system with better cartilage targeting, cartilage penetration and uptake, and joint retention, is needed in large animal models before translating this approach into clinical applications (54).

MATERIALS AND METHODS

Materials

Thioetheramide-PC was purchased from Cayman Chemical (Ann Arbor, MI). HSPC, DSPE-PEG2000, DOTAP, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine rhodamine B sulfonyl) (Rhod-PE), DSPE-PEG2000-Cy7, and NBD-PC were purchased from Avanti Polar Lipids Inc. sPLA2 enzyme from Naja mossambica was purchased from Sigma-Aldrich Co. Rabbit polyclonal antibody to sPLA2-IIa, p-P65, and p-P100 antibodies were purchased from Abcam. Bovine synovial fluid was obtained from Lampire Biological Laboratories Inc. All other chemicals were used as received. All of the buffer solutions were prepared with deionized water.

Synthesis of sPLA2i-loaded phospholipid NPs

sPLA2i-NPs were prepared by hydration of dry sPLA2i/lipid films. A mixture containing 10 mol % DOTAP/25 mol % thioetheramide-PC/65 mol % DSPE-PEG2000 was prepared in a round-bottom flask. The total amount of thioetheramide-PC was 0.25 mg. For the preparation of fluorescently labeled NPs, a small amount (1 mol %) of the fluorescent lipids, Rhod-PE, or DSPE-PEG2000-Cy7, was also added to the thioetheramide-PC/lipid mixture. The solvent was removed using a direct stream of nitrogen before vacuum desiccation for a minimum of 4 hours. NPs were formed by adding deionized water to the dried film and incubating in a 25°C water bath for 5 min and then votexing for another 3 min. The resulting solution was then centrifuged at 3000g for 5 min to remove the large aggregates. Last, the NPs were filtered through a 0.22-μm cellulose acetate membrane filter (Nalgene, Thermo Fisher Scientific) and stored in the dark at 4°C. Ctrl-NPs, including NPs without sPLA2i (10 mol % DOTAP/25 mol % HSPC/65 mol % DSPE-PEG2000) and NPs without DOTAP (10 mol % HSPC/25 mol % sPLA2i/65 mol % DSPE-PEG2000), were prepared using the similar procedures as above.

Synthesized sPLA2i-NPs were incubated in 0.1 M PBS (pH 7.4) and in bovine synovial fluid at 37°C for the stability study. The diameter and zeta potential of the NPs were measured with DLS (Zetasizer, Nano-ZS, Malvern). The morphology of the NPs was observed using a TEM (JOEL 1010) by the negative-staining technique. Phosphorus and sulfur concentration in the sample was quantified by inductively coupled plasma – optical emission spectrometry (ICP-OES) (SPECTRO GENESIS GmbH).

In vitro sPLA2 response study

To study in vitro sPLA2 response of sPLA2i-NPs, the NBD-incorporated liposomes were incubated with sPLA2i-NPs in the presence of sPLA2 enzyme, and the dequenching of NBD fluorescence signal was monitored. To prepare NBD liposomes, stock solutions of HSPC and NBD-PC in chloroform were mixed in the following molar ratios: HSPC/NBD-PC (80:20). The total amount of HSPC was 1 mg. The solvent was removed using a direct stream of nitrogen before vacuum desiccation for a minimum of 4 hours. A total of 0.2 ml of deionized water was then added to the dried lipid film and incubated in a 50°C water bath for 0.5 hours and then sonicated for another 30 min. The stock solution of NBD-incorporated liposomes was stored in the dark at 4°C.

Fluorescence measurements of the NBD-incorporated liposomes in 10 mM Hepes buffer (pH 7.4) [2 mM [CaCl2] and [HSPC] (0.022 mg ml−1)] were taken 10 min before the addition of the Ctrl-NPs, sPLA2i-NPs, or free sPLA2is at different sPLA2i concentrations: 0, 0.125, 0.25, 0.5, 1, and 2 μM. At a time of 20 min, sPLA2 enzyme was added at a final concentration of 0.05 U ml−1. The fluorescence intensity at 520 nm was measured on a SPEX FluoroMax-3 spectrofluorometer (HORIBA Jobin Yvon) using an excitation at 460 nm. The amount of NBD dequenched (% NBD dequenched) was calculated by means of Eq. 1

% NBD dequenched=([Ix−I0]/[It−I0])×100

(1)where I0 is the fluorescence intensity of the liposomal suspension containing NBD at the initial time, Ix is the fluorescence intensity at any given time, and It is the fluorescence intensity after addition of 20 μl of Triton X-100 (50 mM) to the suspension at the end of experiment.

Cell culture

Primary mouse chondrocytes were isolated from the distal femoral and proximal tibial epiphysis of mice (3 to 6 days old) via enzymatic digestion. Briefly, tissues were incubated with 0.25% trypsin (Invitrogen) for 1 hour, followed by 2-hour digestion with type I collagenase (300 U ml−1) (Worthington Biochemical). Dissociated cells were seeded in culture plates, and attached cells were considered primary mouse chondrocytes. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum, streptomycin (100 μg ml−1), and penicillin (100 U ml−1). Primary mouse chondrocytes between passages 0 and 3 were used for MTT assay experiments.

MTT assay

Primary mouse chondrocytes (5000 cells per well) were seeded in 96-well plates and incubated overnight (37°C and 5% CO2) to allow the cells to attach to the surface of the wells. The sPLA2i-NPs were added to wells at five different sPLA2i concentrations ranging from 0.625 to 10 μg ml−1 (0.625, 1.25, 2.5, 5, and 10 μg ml−1), and the cell viabilities were determined according to the supplier’s instructions. After 24 hours of incubation, the medium containing NPs in each well was aspirated off and replaced with 100 μl of fresh medium and 10 μl of MTT reagent. The cells were incubated for 2 to 4 hours. Then, 100 μl of detergent reagent was added and left at room temperature in the dark for 4 hours. Last, the absorbance of formazan product was measured on a Tecan microplate reader (BioTek Instruments Inc.) at 570 nm. Cell viability was calculated using the following equation

Cell viability (%)=(Absorbancesample/Absorbancecontrol)×100

(2)

Bovine cartilage explant harvest and culture

Young (1 to 2 weeks old) bovine knee joints were obtained from vendors (Lampire Biological Laboratories), and cartilage explants were harvested from the trochlear groove using biopsy punch and cultured with chemically defined medium [DMEM, 1% insulin-transferrin-selenous acid (ITS) + premix, l-proline (50 μg ml−1 ), 0.1 μM dexamethasone, 0.9 mM sodium pyruvate, and ascorbate 2-phosphate (50 μg ml−1)] in a 48-well plate.

Bovine cartilage explant penetration assay

Bovine cartilage explants (6 mm in diameter and 2 mm in thickness) were incubated with free rhodamine, rhodamine-labeled sPLA2i-NPs (DOTAP−), or sPLA2i-NPs (DOTAP+) in 500 μl of culture medium for 48, 96, 144, or 192 hours at 37°C and 5% CO2 under gentle agitation. Culture medium with free rhodamine, rhodamine-labeled sPLA2i-NPs (DOTAP−), or sPLA2i-NPs (DOTAP+) was replaced every other day. In all cases, the final rhodamine concentration in the culture medium was 10 μM. After incubation, cartilage explants were washed three times with PBS, fixed with 4% paraformaldehyde (PFA), dehydrated with 20% sucrose + 2% polyvinylpyrrolidone (PVP), followed by embedding with 30% sucrose + 2% PVP + 8% gelatin. Sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount-G mounting medium on glass slides and immediately observed under confocal microscope (Zeiss LSM 710). Quantitative analysis was performed on maximum intensity projections of Z-stack images taken from 100-μm-thick sections.

Bovine cartilage explant uptake assay

A total volume of 300 μl of free rhodamine, rhodamine-labeled sPLA2i-NPs (DOTAP−), or sPLA2i-NPs (DOTAP+) in culture medium was added to bovine cartilage explants (3 mm in diameter and 2 mm in thickness). The final rhodamine concentration in the culture medium was 10 μM. The explants were incubated for 24 hours at 37°C and 5% CO2 under gentle agitation. The explants were then removed from the medium, washed three times with PBS, and imaged by IVIS (Spectrum, PerkinElmer). Radiant efficiency within a fixed anatomical region of interest (ROI) was measured using Living Image software.

In vivo joint retention assay

The mouse knee joints retention assay was assessed by intra-articular injection of 10 μl of 10 μM free ICG or 10 μM Cy7-doped sPLA2i-NPs in healthy (3 months old) and OA (8 weeks after DMM surgery) mouse knees. The rat knee joints retention assay was assessed by intra-articular injection of 40 μl of 10 μM free ICG or 10 μM Cy7-doped sPLA2i-NPs in healthy rat knees (3 months old). An IVIS (Spectrum, PerkinElmer) was used to serially acquire fluorescence images within each joint over a period of 4 weeks. Using Living Image software, radiant efficiency within a fixed anatomical ROI was measured.

In vivo biodistribution assay

In vivo biodistribution study was performed by intra-articular injection of 10 μl of PBS or 10 μM Cy7-doped sPLA2i-NPs in mouse knees (3 months old). At 24 hours or 1 month following injection, the mice were euthanized. The knee joints, blood, and major organs (heart, liver, spleen, lung, and kidney) were harvested. Knees were dissected to isolate the major joint components, including the surrounding tissues (quadriceps, patella, patellar ligament, synovium, and fat pad), femoral condyles, tibial plateau, and meniscus. All the major joint components, blood, and organs were imaged using the IVIS with the following imaging parameters: excitation, 745 nm; emission, 800 nm; exposure time, 1 s. The data were analyzed as described above.

Mouse femoral head explants penetration assay

Mouse femoral heads were collected from 8-week-old male mice and cultured for 48 hours in chemically defined medium [DMEM, streptomycin (100 μg ml−1) and penicillin (100 U ml−1), 1% ITS + premix, l-proline (40 μg ml−1), 0.1 μM dexamethasone, sodium pyruvate (100 μg ml−1), and ascorbate 2-phosphate (50 μg ml−1)] at 37°C and 5% CO2 in a 48-well plate. Following the culture, mouse femoral head explants were stimulated using recombinant mouse IL-1β (10 ng ml−1; PeproTech) for 2 days. On day 3, rhodamine-labeled sPLA2i-NPs were incubated with the IL-1β–stimulated femoral heads for 24 or 48 hours at 37°C with gentle agitation. The final rhodamine concentration in the culture medium was 10 μM. Following incubation, femoral heads were washed with PBS, fixed with 4% PFA, dehydrated with 30% sucrose, and embedded in Tissue-Tek optimum cutting temperature compound. The 6-μm-thick cyrosections were cut and mounted with DAPI Fluoromount-G mounting medium on the slides and imaged with a fluorescence microscope (Nikon, Eclipse 90i). Images were analyzed with ImageJ to quantify the penetration depth NP into the cartilage. Fluorescence intensity within each image was measured with ImageJ and normalized to the fluorescence of the outermost cartilage surface of the treated femoral head.

Mouse femoral head explants OA degradation assay

Mouse femoral heads were collected using the same procedure as described in mouse femoral head explants penetration assay. To test the therapeutic effects of sPLA2i-NPs, femoral head cartilage explants were divided into five groups to receive PBS (i.e., untreated), IL-1β, IL-1β and Ctrl-NP, IL-1β and sPLA2i, and IL-1β and sPLA2i-NP treatments, respectively, for 8 days. The final IL-1β and sPLA2i concentration in the culture medium was 10 ng ml−1 and 0.1 mg ml−1, respectively. The culture medium was replaced at days 2, 4, and 6. After an 8-day incubation, mouse femoral heads were then fixed with 4% PFA overnight, followed by decalcification in 0.5 M EDTA (pH 7.4) for 2 weeks and processed for 6-μm paraffin sections. Paraffin sections were used for Safranin O staining, and the Safranin O–positive area was quantified with ImageJ. OA severity was evaluated by Mankin score. Both quantifications were based on the section with the most severe loss of Safranin O staining and cartilage damage.

Quantitative reverse transcription polymerase chain reaction

Mouse femoral heads were collected and treated using the same procedure in femoral head explants degradation assay. Total RNA was harvested from the femoral head articular cartilage using TRI Reagent (Sigma-Aldrich Co.). TaqMan Reverse Transcription kit (Applied Biosystems) was used to reverse-transcribe mRNA into complementary DNA. Following this, polymerase chain reaction (PCR) was performed using a Power SYBR Green PCR Master Mix kit (Applied Biosystems). The primer sequences for the genes used in this study are listed in table S1.

Human articular cartilage samples

The human healthy and OA articular cartilage samples were prepared from the deidentified specimens obtained at the total arthroplasty of the knee joints and processed for paraffin sections. The serial sections were stained by Safranin O and Fast Green staining to evaluate OA grades as previously described (55). For sPLA2 staining, anti–sPLA2-IIA (1:800; Abcam, ab23705) antibody was used. We considered healthy, early OA, and middle OA cartilage as grades 0, 1 to 2, and 3 to 4 based on Osteoarthritis Research Society International score, respectively.

Animal care

In accordance with the standards for animal housing, mice with C57BL/6 background were group-housed in an atmosphere of 23° to 25°C with a 12-hour light/dark cycle and allowed free access to water and standard laboratory pellets. All work performed on animals was approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

To induce mouse OA that mimics chronic OA in human patients, male mice at 3 months of age were subjected to DMM surgery at right knees and sham surgery at left knees. Briefly, in DMM surgery, the joint capsule was opened immediately after anesthesia, and the medial meniscotibial ligament was cut to destabilize the meniscus without damaging other tissues. In sham surgery, the joint capsule was opened in the same fashion but without any further damage.

To induce mouse OA that is noninvasive and mimics PTOA in human patients, male mice (2 months old) were subjected to mechanic loading at the right knees and sham loading at left knees. Briefly, under anesthesia, the right tibiae were positioned with the knee downward in deep flexion between custom-made cups and subjected to axial compressive loads with a peak force of 6 or 9 N with a 0.5-N preload force to maintain the limb in position between loading cycles. Cyclic loads were applied for 0.34 s with a rise and fall time each of 0.17 s and a baseline hold time of 10 s between cycles for 60 cycles. The uninjured left knees were used as controls.

NP administration

For treatment, NPs were administrated using sterile techniques: The right knees were kept in a flexed position, and a total volume of 10 μl of PBS, sPLA2i (0.25 mg ml−1), Ctrl-NPs, or sPLA2i-NPs (sPLA2i, 0.25 mg ml−1) was injected intra-articularly with a 30-gauge needle. For DMM surgical OA model, the first injection was performed immediately after surgery. Injections were then repeated every 2 weeks for 2 or 4 months. In total, there are four injections for the 2-month group and eight injections for 4-month group. For load-induced OA model, intra-articular injections were performed immediately and at 48 hours after loading. There are two injections in total for each mouse in this model.

After 2-month treatment for DMM mice, some major organs (kidney, liver, lung, heart, and spleen) and blood were collected. Tissue sections were stained with hematoxylin and eosin (H&E) to assess the effects of different treatments on mouse organ morphology. The blood indexes were measured after receiving 2-month treatment of PBS, sPLA2i, Ctrl-NPs, or sPLA2i-NPs.

Histology

After euthanasia, mouse knee joints were harvested and fixed in 4% PFA overnight, followed by decalcification in 0.5 M EDTA (pH 7.4) for 4 weeks before paraffin embedding. A serial of 6-μm-thick sagittal sections (about 100) were cut across the entire medial compartment of the joint until ACL junction. To measure the thicknesses of articular cartilage and chondrocyte numbers, three sections from each knee, corresponding to one of four (sections 20 to 30), two of four (sections 45 to 55), and three of four (sections 70 to 80) regions of the entire section set, were stained with Safranin O/Fast Green and quantified using BIOQUANT software. The final measurement is an average of these three sections. We defined uncalcified cartilage as the area from articular surface to the tide mark and calcified cartilage as the area from tide mark to cement line. The method to measure Mankin score was described previously (56). Briefly, two sections within every consecutive six sections in the entire section set for each knee were stained with Safranin O/Fast Green and scored by two blinded observers. Each knee received a single score representing the maximal score of its sections.

Synovitis score grading was carried out in 6-μm paraffin sections of sagittal mouse knee sections stained with H&E. The following basic morphological parameters of synovitis were included: (i) hyperplasia/enlargement of synovial lining layer, (ii) degree of inflammatory infiltration, and (iii) activation of resident cells/synovial stroma, including fibroblasts, endothelial cells, histiocytes, macrophages, and multinucleated giant cells. All parameters are graded from 0 (absent), 1 (slight), 2 (moderate) to 3 (strong positive).

Cartilage injury length was measured in Safranin O/Fast Green–stained paraffin sections. From the serial Safranin O–stained sections in each sample, we selected one section with the widest cartilage lesion that featured by focal loss of Safranin O staining, minor fissuring of the articular cartilage, and atrophy of articular chondrocytes. According to these histological changes, it is possible to identify the demarcation between the normal and injured cartilage tissue, and the length of the cartilage lesion range was measured.

Paraffin sections were used for IHC and TUNEL assay. For mouse samples, after appropriate antigen retrieval, slides were incubated with primary antibodies, such as rabbit anti–sPLA2-IIA (1:100; Abcam, ab23705), rabbit anti-p-P65 (1:50: Abcam, ab86299), and rabbit anti-p-P100 (1:50; Abcam, ab194919) at 4°C overnight, followed by binding with biotinylated secondary antibodies and DAB (3,3′-diaminobenzidine) color development. The TUNEL assay was carried out according to the manufacturer’s instructions (Millipore, s7101).

OA pain analysis

The knee joint pain after DMM surgery or loading injury was evaluated in mice weekly before and after surgery using von Frey filaments as described previously (57). An individual mouse was placed on a wire-mesh platform (Excellent Technology Co.) under a 4 cm by 3 cm by 7 cm cage to restrict their move. Mice were trained to be accustomed to this condition every day starting from 7 days before the test. During the test, a set of von Frey fibers (Stoelting Touch Test Sensory Evaluator Kit #2 to #9, ranging from 0.015- to 1.3-g force) were applied to the plantar surface of the hind paw until the fibers bowed and then held for 3 s. The threshold force required to elicit withdrawal of the paw (median, 50% withdrawal) was determined five times on each hind paw with sequential measurements separated by at least 5 min.

Micro–computed tomography analysis

The distal femur of mouse knee joints was scanned at a 6-μm isotropic voxel size with a micro–computed tomography (micro-CT) 35 scanner (Scanco Medical AG, Brüttisellen, Switzerland). All images were smoothened by a Gaussian filter (sigma = 1.2, support = 2.0). Measurement of SBP thickness was described previously (58). Briefly, sagittal images were contoured for the SBP, followed by generating a 3D color map of thickness for the entire SBP along with a scale bar. This map was then converted to a grayscale thickness map. The ROI was circled, and the average SBP thickness within ROI is calculated by average gray value/255 × maximum scale bar value. Coronal images were also contoured for the osteophyte, followed by 3D reconstruction and volume calculation.

Statistical analysis

Data are expressed as means ± SEM and analyzed by t tests, one-way analysis of variance (ANOVA) with Dunnett’s or Turkey’s post test, and two-way ANOVA with Bonferroni’s or Turkey’s post test for multiple comparisons using Prism 8 software (GraphPad Software, San Diego, CA). For assays using primary chondrocytes and bovine cartilage explants, experiments were repeated independently at least three times, and representative data were shown here. Values of P < 0.05 were considered statistically significant.

Acknowledgments: Funding: This study was supported by NIH grants NIH/NIAMS R01AG067698 (to L.Q.), P30AR069619 (to Penn Center for Musculoskeletal Disorders), R01NS100892 (to Z.C.), and 2019 Health Research Formula Fund from the Pennsylvania Department of Health (to Z.C.). Author contributions: L.Q. and Z.C. designed the study. Y.W., L.L., and T.G. performed animal experiments. L.L., A.A., and L.Y. made NP formulations. Y.W., L.L., L.Y., and T.G. performed the histology and imaging analysis. Y.W. and T.G. performed cell culture and quantitative reverse transcription PCR experiments. B.J. performed the ICP-OES measurement. T.Y. and A.T. provided technical or material support and consultation. L.Q. and Z.C. wrote the manuscript. A.T. reviewed and revised the manuscript. L.Q. and Z.C. approved the final version. Competing interests: Z.C., L.Q., A.T., and Y.W. are inventors on a patent related to this work filed by the University of Pennsylvania (no. 63/043,025., filed 23 June 2020). The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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[healthtimetaylor]

Absence of gut microbiota affects lipid metabolism in the prefrontal cortex of mice.

PMID:  Neurol Res. 2019 Oct 7:1-9. Epub 2019 Oct 7. PMID: 31587617 Abstract Title:  Absence of gut microbiota affects lipid metabolism in the prefrontal cortex of mice. Abstract:  : Lipid metabolism is closely associated with many important biological functions. Here, we conducted this study to explore the effects of gut microbiota on the lipid metabolism in the prefrontal cortex of mice.: Germ-free (GF) mice, specific pathogen-free (SPF) and colonized GF (CGF) mice were used in this study. The open field test (OFT), forced swimming test (FST) and novelty suppressed feeding test (NSFT) were conducted to assess the changes in general behavioral activity. The liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was used to obtain the lipid metabolites. Both one-way analysis of variance (one-way ANOVA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to obtain the key differential lipid metabolites.: The behavioral tests showed that compared to SPF mice, GF mice had more center distance, more center time, less immobility time and less latency to familiar food. Meanwhile, 142 key differential lipid metabolites between SPF mice and GF mice were identified. These lipid metabolites mainly belonged to glycerophospholipids, glycerolipids, sphingolipids, and saccharolipids. The gut microbiota colonization did not reverse these changed behavioral phenotypes, but could restore 25 key differential lipid metabolites.: These results showed that the absence of gut microbiota could influence host behaviors and lipid metabolism. Our findings could provide original and valuable data for future studies to further investigate the microbiota-gut-brain axis.

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[its-lifestyle]

Adults consuming wholegrain rye have lower plasma serotonin levels than people eating low-fibre wheat bread, according to a recent study by the University of Eastern Finland and the International Agency for Research on Cancer (IARC).

In the study, the consumption of cereal fibre from rye or wheat was also found to reduce serotonin levels in the colon of mice.

In light of the results, the health benefits of wholegrain cereals may be linked, at least in part, to the alteration of serotonin production in the intestines, where the majority of the body’s serotonin is produced.

The results were published in the American Journal of Clinical Nutrition on April 26, 2019.

The consumption of wholegrain cereals has been associated with a lower risk of type 2 diabetes, cardiovascular disease and some cancers, but the underlying mechanisms are still poorly understood.

There may be effects on bioactive compounds contained in whole grains, phytochemicals and fibres, from which different metabolites are produced by intestinal bacteria.

The new study explored how the consumption of wholegrain rye modulates concentrations of different metabolites in the bloodstream.

The study employed untargeted metabolite profiling, also known as metabolomics, which can simultaneously detect numerous metabolites, including those previously unknown.

For the first four weeks of the study, the participants ate six to 10 slices a day of low-fibre wheat bread, and then, the same amount of wholegrain rye bread or wheat bread supplemented with rye fibre for another four weeks.

Otherwise, they didn’t change their diet.

At the end of both periods, they gave blood samples, which were analysed by a combination of liquid chromatography and mass spectrometry.

Their plasma metabolite profiles between the different diet periods were then compared.

The consumption of wholegrain rye led to, among other things, significantly lower serotonin concentrations, when compared to consumption of low-fibre wheat bread.

The researchers also tested in mice whether the addition of cereal fibre to the diet changes serotonin production in the intestine.

The diet of the mice was supplemented for nine weeks with rye bran, wheat bran or cellulose flour.

The mice receiving rye or wheat bran had significantly lower serotonin in their colon.

Serotonin is best known as a neurotransmitter in the brain.

However, serotonin produced by the intestines remains separated from the brain, serving various peripheral functions including modulation of the gut’s motility.

Increased blood serotonin has also been associated with high blood glucose levels.

“Wholegrain, on the other hand, is known to reduce the risk of diabetes, and on the basis of these new results, the effect could at least partly be due to a decrease in serotonin levels,” says Academy Research Fellow Kati Hanhineva from the University of Eastern Finland.

The researchers are also interested in the association of serotonin with colorectal cancer.

“Some recent studies have found cancer patients have higher plasma serotonin levels than healthy controls,” IARC scientist Pekka Keski-Rahkonen adds.

The consumption of wholegrain rye bread was also associated with lower plasma concentrations of taurine, glycerophosphocholine and two endogenous glycerophospholipids.

In addition, the researchers identified 15 rye phytochemicals whose levels in the bloodstream increased with the consumption of rye fibre.

from Food – Star2.com http://bit.ly/2JUErPR

[rnomics]

Microorganisms, Vol. 7, Pages 358: A Discovery of Relevant Hepatoprotective Effects and Underlying Mechanisms of Dietary Clostridium butyricum Against Corticosterone-Induced Liver Injury in Pekin Ducks

Clostridium butyricum (C. butyricum) can attenuate oxidative stress, inflammation, and hepatic fatty deposition in poultry, however, the underlying mechanisms for this in Pekin ducks remain unclear. This study evaluated these hepatoprotective effects and the underlying mechanisms in a corticosterone (CORT)-induced liver injury model in Pekin ducks fed a C. butyricum intervention diet. A total of 500 Pekin ducks were randomly divided into five groups: one group (CON group) was only provided with a basal diet, three groups were provided a basal diet with 200 mg/kg (LCB group), 400 mg/kg (MCB group), or 600 mg/kg (HCB group) C. butyricum, respectively, and one group was provided a basal diet with 150 mg/kg aureomycin (ANT group) for 42 d. At 37 days-old, all ducks received daily intraperitoneal injections of CORT for five days to establish a liver injury model. C. butyricum intervention alleviated liver injury by decreasing the liver organ indices, hepatic steatosis and hepatocyte necrosis, and improving liver function, antioxidant capacity, and inflammatory factors. Hepatic #RNA-seq revealed 365 differentially expressed genes (DEGs) between the MCB and CON groups, with 229 up- and 136 down-regulated DEGs in the MCB group. Between the MCB and ANT groups, 407 DEGs were identified, including 299 up- and 108 down-regulated genes in MCB group. Some DEGs in the MCB group related to oxidative stress and inflammatory responses such as Sod3, Tlr2a/b, and Il10, which were up-regulated, while Apoa1, Cyp7a1, Acsl1/5, Fasn, Ppar-&gamma;, and Scd, which are involved in lipid metabolism, were down-regulated, indicating that these genes were responsive to dietary C. butyricum for the alleviation of corticosterone-induced hepatic injury. Toll-like receptor signaling, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, peroxisome proliferator-activated receptor (PPAR) signaling pathway, adipocytokine and glycerophospholipid metabolism signaling pathway were significantly enriched in the MCB group. These findings indicate that C. butyricum intervention can protect Pekin ducks from corticosterone-induced liver injury by the modulation of immunoregulatory- and lipid metabolism-related genes and pathways. http://bit.ly/2O4HeIk

[abbkineeu]

New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/anti-virus-monoclonal-antibody-could-be-elicited-more-efficiently-with-suitably-optimized-gp-immunogens/

Anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens

Topics overview: Dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity, mechanisms controlling immunity to vaccination, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism, CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading and anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, Nina Cabezas-Wallscheid at Division of Stem Cells and Cancer in Heidelberg, Germany and her colleagues use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Their results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30464-6

  Metabolic Phenotypes of Response to Vaccination in Humans.

Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, Shuzhao Li at Emory University in Atlanta, USA and his colleagues constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Their approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30477-4

  Widespread Influence of 3′-End Structures on Mammalian mRNA Processing and Stability.

The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, Xuebing Wu at Howard Hughes Medical Institute and Whitehead Institute for Biomedical Research in Cambridge, USA and his colleagues investigate the possibility that mRNA structures facilitate the 3′-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. They find that RNA structures are predicted to be more prevalent within these extended 3′-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30490-7

  Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, Elphège P. Nora at Gladstone Institute of Cardiovascular Disease in San Francisco, USA and his colleagues show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, their data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30531-7

  Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, Anna Z. Wec at Department of Microbiology and Immunology, Albert Einstein College of Medicine in Bronx, NY , USA and her colleagues mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Their findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30491-9

[bioadvisers]

New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/anti-virus-monoclonal-antibody-could-be-elicited-more-efficiently-with-suitably-optimized-gp-immunogens/

Anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens

Topics overview: Dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity, mechanisms controlling immunity to vaccination, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism, CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading and anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, Nina Cabezas-Wallscheid at Division of Stem Cells and Cancer in Heidelberg, Germany and her colleagues use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Their results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30464-6

  Metabolic Phenotypes of Response to Vaccination in Humans.

Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, Shuzhao Li at Emory University in Atlanta, USA and his colleagues constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Their approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30477-4

  Widespread Influence of 3′-End Structures on Mammalian mRNA Processing and Stability.

The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, Xuebing Wu at Howard Hughes Medical Institute and Whitehead Institute for Biomedical Research in Cambridge, USA and his colleagues investigate the possibility that mRNA structures facilitate the 3′-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. They find that RNA structures are predicted to be more prevalent within these extended 3′-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30490-7

  Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, Elphège P. Nora at Gladstone Institute of Cardiovascular Disease in San Francisco, USA and his colleagues show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, their data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30531-7

  Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, Anna Z. Wec at Department of Microbiology and Immunology, Albert Einstein College of Medicine in Bronx, NY , USA and her colleagues mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Their findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30491-9

[abbkine]

New Post has been published on Biotech Advisers

Anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens

Topics overview: Dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity, mechanisms controlling immunity to vaccination, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism, CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading and anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, Nina Cabezas-Wallscheid at Division of Stem Cells and Cancer in Heidelberg, Germany and her colleagues use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Their results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30464-6

  Metabolic Phenotypes of Response to Vaccination in Humans.

Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, Shuzhao Li at Emory University in Atlanta, USA and his colleagues constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Their approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30477-4

  Widespread Influence of 3′-End Structures on Mammalian mRNA Processing and Stability.

The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, Xuebing Wu at Howard Hughes Medical Institute and Whitehead Institute for Biomedical Research in Cambridge, USA and his colleagues investigate the possibility that mRNA structures facilitate the 3′-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. They find that RNA structures are predicted to be more prevalent within these extended 3′-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30490-7

  Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, Elphège P. Nora at Gladstone Institute of Cardiovascular Disease in San Francisco, USA and his colleagues show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, their data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30531-7

  Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, Anna Z. Wec at Department of Microbiology and Immunology, Albert Einstein College of Medicine in Bronx, NY , USA and her colleagues mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Their findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30491-9

[abbkineus]

New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/anti-virus-monoclonal-antibody-could-be-elicited-more-efficiently-with-suitably-optimized-gp-immunogens/

Anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens

Topics overview: Dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity, mechanisms controlling immunity to vaccination, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism, CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading and anti-virus monoclonal antibody could be elicited more efficiently with suitably optimized GP immunogens.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, Nina Cabezas-Wallscheid at Division of Stem Cells and Cancer in Heidelberg, Germany and her colleagues use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Their results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30464-6

  Metabolic Phenotypes of Response to Vaccination in Humans.

Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, Shuzhao Li at Emory University in Atlanta, USA and his colleagues constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Their approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30477-4

  Widespread Influence of 3′-End Structures on Mammalian mRNA Processing and Stability.

The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, Xuebing Wu at Howard Hughes Medical Institute and Whitehead Institute for Biomedical Research in Cambridge, USA and his colleagues investigate the possibility that mRNA structures facilitate the 3′-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. They find that RNA structures are predicted to be more prevalent within these extended 3′-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30490-7

  Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, Elphège P. Nora at Gladstone Institute of Cardiovascular Disease in San Francisco, USA and his colleagues show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, their data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30531-7

  Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, Anna Z. Wec at Department of Microbiology and Immunology, Albert Einstein College of Medicine in Bronx, NY , USA and her colleagues mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Their findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Read more, please click http://www.cell.com/cell/fulltext/S0092-8674(17)30491-9

[heilpraktiker-hannover]

Hot Chilies Can Cool Gut Inflammation

Hot Chilies Can Cool Gut Inflammation

Hot Chilies Can Cool Gut Inflammation

by Dr. Mercola, articles.mercola.comMay 15, 2017

Research has recently discovered that hot chilies may help soothe your gut when you suffer from inflammatory bowel disease (IBD). IBD is not to be confused with inflammatory bowel syndrome (IBS), which is a less serious functional disorder that doesn't cause ulcers or intestinal damage as does IBD.

IBD is an autoimmune condition that may have serious consequences. Currently there are nearly 3 million Americans who suffer from IBD, with nearly 70,000 new cases diagnosed each year.1 This is nearly triple previous estimates, which may be due to a combination of rising rates and improved diagnostic criteria.

The rising rates of the condition may be related to genetics, environmental factors, diet and changes in your gut microbiome. Research has demonstrated your microbiome is so important that physicians may even use it as a diagnostic tool.2

While improving your microbiome plays a significant role in reducing or eliminating your symptoms, capsaicin and endocannabinoids may also play a role in reducing your symptoms through control of your neuroimmune axis.3 Before understanding how it works, it's helpful to understand the condition.

What Is Inflammatory Bowel Disease?

IBD is a chronic inflammation of part or all of your intestinal tract that may result in ulcerations, vomiting, bloody diarrhea and weight loss. The condition is often debilitating and may lead to a life-threatening situation. There are two different types of IBD: Crohn's disease and ulcerative colitis (UC).

UC is generally more common in men, while more women suffer from Crohn's disease.4 Both forms of IBD are more common in developed countries, while ex-smokers and nonsmokers more commonly have UC, and smokers more commonly develop Crohn's.

People who experience UC will often suffer from ulcers and inflammation in the lining of the large intestines, while Crohn's is a disease that affects your entire digestive tract. Doctors believe your symptoms are the result of widespread recurring immune response focused in your intestines.5

Your immune system mistakes substances in your intestines for a foreign material and sends out white blood cells that trigger inflammation.6 Without significant changes to your lifestyle, symptoms continue to get worse and affect your quality of life.

Unfortunately, the symptoms are often associated with stigma, fear and isolation that have resulted in the disease being relatively hidden and people suffering in silence. While there is no real cure for the condition, there are strategies that may reduce or eliminate your symptoms.

Why Hot Chilies May Calm Your Gut

Recent research has found when mice were fed capsaicin, the substance in chili peppers that makes them "hot," they had less inflammation in their gut — and some were even cured of a mouse model type 1 diabetes.7 Researchers found capsaicin acted on receptors, causing local production of anandamide.

Anandamide acts through your cannabinoid receptor 2 (CB2) that increases the number and function of immune macrophages in your gut. You have endogenous cannabinoids or endocannabinoids produced in your system from glycerophospholipids. Anadamide is an endogenous intestinal cannabinoid.

It controls your appetite and energy balance through the nervous system in your intestinal tract. This study uncovered the immunological role anandamide and endocannabinoids play in the regulation of immune tolerance in the gut,8 and in maintaining immune homeostasis between the nervous and immune systems.

Following the discovery of endogenous cannabinoid production in the mid-2000s, a large number of studies exploring the system and regulatory function were initiated, as scientists were hopeful for a pharmaceutical approach to health.9

Interactions in the endocannabinoid system are much like those that occur in the brain, as your gut has a very large nervous system affected by the endocannabinoids produced after ingesting chili peppers. Researchers question if people who use edible cannabinoids may experience the same relief.10

What Is Cannabidiol or CBD?

The cannabis plant, commonly known as marijuana, has two major active ingredients. The more famous molecule is tetrahydrocannabinol (THC), known for the psychoactive properties that leave people feeling "high" or "stoned."11 The second chemical compound found in the cannabis plant is cannabidiol (CBD). This doesn't have the psychoactive effects but still has an effect on your body. CBD may actually counteract the psychoactivity of THC.12

How much THC and CBD in the plant depends upon the strain. In the past decade much research has been undertaken to ascertain the medical efficacy of CBD in the treatment of specific conditions. There has been promising evidence of positive effects as an antidepressant,13 anti-inflammatory,14 antioxidant15 and neuroprotective.16

Often used in oil form, medicinal cannabis oil is extracted from strains of the plant grown specifically high in CBD and low in THC. CBD works by binding to cannabinoid receptor cells, some heavily concentrated in your nervous system. However, there are receptors in almost every organ of your body.17

The combination of your endogenous cannabinoid and receptors make up your endocannabinoid system, involved in many of your organ systems. However, the four primary purposes appear to be neuroprotection, immune balance, stress recovery and homeostasis regulation.18

Although both THC and CBD are cannabinoids, they react with your body in different ways. THC engages your CBD1 receptors, many of which are in your central nervous system, or brain. Combined with the psychoactive properties, this results in feeling "high."19

CBD Results Push Further Research

Neurologist Dr. Ethan Russo discussed the difference between THC and CBD and the way they interact with your receptors in an interview with Project CBD. He points out that cannabis has been selectively bred over the years for high THC levels to increase the psychoactive effects of the plant.20

However, the medicinal plant bred for high levels of CBD and low levels of THC do not engage your CBD1 receptors in the same way. He explains:

"It doesn't tend to bind directly, what's called the orthosteric site where THC binds. Rather, it binds on what's called an allosteric site, another site on the receptor, and it so it alters the binding of both THC and the endogenous cannabinoids, the endocannabinoids.

So, cannabidiol is what's called the negative allosteric modulator, which is a fancy way of saying that when THC is present it interferes with its activity — which is a good thing in terms of wanting too much psychoactivity and again limiting side effects like anxiety or rapid heart rate that can be a problem if someone has too much THC."

This difference has fueled a group of "medical cannabis refugees," who have moved to Colorado in search of medicinal plants that have demonstrated unique and powerful success in the treatment of conditions like intractable seizures,21 Parkinson's disease22,23 and obsessive compulsive disorder.24,25

In the past decade the endocannabinoid system has been implicated in a number of biological and physiological functions that show increasing promise in treatment of a wide variety of disorders, including IBD, neuropathic pain and multiple sclerosis.26

However, while great strides have been made in research, the socially unacceptable psychoactive properties of the cannabis plant have presented a significant challenge to families seeking treatment. The use of selective receptor agonists could potentially improve treatment options.

How CBD May Affect Your Gut Health

Research from the University of Vermont found those who ate red-hot chili peppers experienced a 13 percent reduction in risk of death, primarily from heart disease or stroke.27 Data suggested participants benefited from the result of capsaicin cellular and molecular mechanisms, especially in the gut.

The outcome of an excessive inflammatory response in your gut is intestinal damage and disturbances in motility and secretion — the hallmarks of IBD.28 With no known current pharmaceutical cure, success of treatment modalities is measured by severity of symptoms.

Active inflammation in the gut may be mediated by enteric glial cells that release factors, amplifying the immune response.29 This represents an important connection between your nervous system and immune system inside your gut. CBD can control these glial cells in your nervous system without psychoactivity, and therefore reduce the inflammatory response in your intestinal tract. These results indicate that CBD meets criteria for a therapeutic strategy to treat IBD.30

The gut-brain connection offers a strong explanation of how your intestinal health responds to stress and, conversely, why acute or chronic abdominal distress may result in emotional complaints. CBD is becoming more recognized for the important role it plays in treating this neuroimmune axis.31

And finally, the side-effect profile for CBD oil and other medicinal preparations of CBD is very low. In fact, the preparations have fewer side effects than current medications used to treat UC and Crohn's disease.32,33

Prevention and Treatment Without Drugs

See Video: http://www.youtube.com/watch?v=XuktG-Z1a84

Like most health conditions, prevention is the best medicine. Your gut bacteria have a powerful influence over your immune system and the inflammatory response in your body as I discuss in the video above.34,35 Individuals who suffer from IBD often share an altered microbiome resulting in a protracted state of inflammation and symptoms.36 Reseeding your gut to achieve a more normal composition should be high on your list to reduce your symptoms or prevent the condition.

Traditionally fermented and unpasteurized cultured foods are loaded with healthy bacteria, and are easy to make from scratch at home. To learn more about fermenting vegetables, see my previous article, "Tips for Fermenting at Home." Other helpful foods that are important if you struggle with IBD include:

• Blueberries. These little berries are packed with antioxidants, vitamins and fiber, and research has demonstrated they may help alleviate and protect against inflammation in your gut.37,38 They are rich in polyphenols that have both antimicrobial and antioxidant effects.

When combined with probiotics, blueberries may reduce inflammatory-inducing bacteria in your gut and increase the amount of healthy Lactobacilla. The fiber doesn't degrade in your large intestines, protecting your intestinal lining from substances that cause inflammation.

• Coconut oil. This is a wonderful overall addition to your diet and has anti-inflammatory effects that may help heal injury to your digestive tract.39Coconut oil also has antimicrobial properties that may kill microorganisms, including yeast that may cause chronic inflammation.

• Caprylic acid. This is a medium-chain triglyceride (MCT oil) that is also a potent antifungal. Dr. Leon Chaitow, author of "Candida Albicans: Could Yeast Be Your Problem?" recommends it in lieu of antifungal drugs for Candida overgrowth.

• Animal-based omega-3. This is another essential element for preventing and controlling IBD. The omega-3 fats in krill oil, EPA and DHA, have immune-boosting qualities along with anti-inflammatory properties proven to benefit disorders of the gut, including Crohn's disease and UC.40

• Fiber-rich foods. Fiber foods such as organic psyllium seed husk, flax, hemp and berries, vegetables such as broccoli and Brussels sprouts, root vegetables and tubers, raw nuts and beans, help nourish beneficial microbes in your gut.

Aim for 50 grams of fiber per 1,000 calories consumed. Supplementing with organic psyllium husk can help bring you closer to this ideal amount. Just make sure that the psyllium is organic, as non-organic psyllium is typically severely contaminated with pesticides.

Original Page: http://ift.tt/2r7FO4V

Shared from Pocket

Tags: -newsletter, -podcast, -ideas, ibd, entz��ndliche darmerkrankung, Chili, -video May 15, 2017 at 02:55PM Open in Evernote

[sherristockman]

Hot Chilies Can Cool Gut Inflammation Dr. Mercola By Dr. Mercola Research has recently discovered that hot chilies may help soothe your gut when you suffer from inflammatory bowel disease (IBD). IBD is not to be confused with inflammatory bowel syndrome (IBS), which is a less serious functional disorder that doesn't cause ulcers or intestinal damage as does IBD. IBD is an autoimmune condition that may have serious consequences. Currently there are nearly 3 million Americans who suffer from IBD, with nearly 70,000 new cases diagnosed each year.1 This is nearly triple previous estimates, which may be due to a combination of rising rates and improved diagnostic criteria. The rising rates of the condition may be related to genetics, environmental factors, diet and changes in your gut microbiome. Research has demonstrated your microbiome is so important that physicians may even use it as a diagnostic tool.2 While improving your microbiome plays a significant role in reducing or eliminating your symptoms, capsaicin and endocannabinoids may also play a role in reducing your symptoms through control of your neuroimmune axis.3 Before understanding how it works, it's helpful to understand the condition. What Is Inflammatory Bowel Disease? IBD is a chronic inflammation of part or all of your intestinal tract that may result in ulcerations, vomiting, bloody diarrhea and weight loss. The condition is often debilitating and may lead to a life-threatening situation. There are two different types of IBD: Crohn's disease and ulcerative colitis (UC). UC is generally more common in men, while more women suffer from Crohn's disease.4 Both forms of IBD are more common in developed countries, while ex-smokers and nonsmokers more commonly have UC, and smokers more commonly develop Crohn's. People who experience UC will often suffer from ulcers and inflammation in the lining of the large intestines, while Crohn's is a disease that affects your entire digestive tract. Doctors believe your symptoms are the result of widespread recurring immune response focused in your intestines.5 Your immune system mistakes substances in your intestines for a foreign material and sends out white blood cells that trigger inflammation.6 Without significant changes to your lifestyle, symptoms continue to get worse and affect your quality of life. Unfortunately, the symptoms are often associated with stigma, fear and isolation that have resulted in the disease being relatively hidden and people suffering in silence. While there is no real cure for the condition, there are strategies that may reduce or eliminate your symptoms. Why Hot Chilies May Calm Your Gut Recent research has found when mice were fed capsaicin, the substance in chili peppers that makes them "hot," they had less inflammation in their gut — and some were even cured of a mouse model type 1 diabetes.7 Researchers found capsaicin acted on receptors, causing local production of anandamide. Anandamide acts through your cannabinoid receptor 2 (CB2) that increases the number and function of immune macrophages in your gut. You have endogenous cannabinoids or endocannabinoids produced in your system from glycerophospholipids. Anadamide is an endogenous intestinal cannabinoid. It controls your appetite and energy balance through the nervous system in your intestinal tract. This study uncovered the immunological role anandamide and endocannabinoids play in the regulation of immune tolerance in the gut,8 and in maintaining immune homeostasis between the nervous and immune systems. Following the discovery of endogenous cannabinoid production in the mid-2000s, a large number of studies exploring the system and regulatory function were initiated, as scientists were hopeful for a pharmaceutical approach to health.9 Interactions in the endocannabinoid system are much like those that occur in the brain, as your gut has a very large nervous system affected by the endocannabinoids produced after ingesting chili peppers. Researchers question if people who use edible cannabinoids may experience the same relief.10 What Is Cannabidiol or CBD? The cannabis plant, commonly known as marijuana, has two major active ingredients. The more famous molecule is tetrahydrocannabinol (THC), known for the psychoactive properties that leave people feeling "high" or "stoned."11 The second chemical compound found in the cannabis plant is cannabidiol (CBD). This doesn't have the psychoactive effects but still has an effect on your body. CBD may actually counteract the psychoactivity of THC.12 How much THC and CBD in the plant depends upon the strain. In the past decade much research has been undertaken to ascertain the medical efficacy of CBD in the treatment of specific conditions. There has been promising evidence of positive effects as an antidepressant,13 anti-inflammatory,14 antioxidant15 and neuroprotective.16 Often used in oil form, medicinal cannabis oil is extracted from strains of the plant grown specifically high in CBD and low in THC. CBD works by binding to cannabinoid receptor cells, some heavily concentrated in your nervous system. However, there are receptors in almost every organ of your body.17 The combination of your endogenous cannabinoid and receptors make up your endocannabinoid system, involved in many of your organ systems. However, the four primary purposes appear to be neuroprotection, immune balance, stress recovery and homeostasis regulation.18 Although both THC and CBD are cannabinoids, they react with your body in different ways. THC engages your CBD1 receptors, many of which are in your central nervous system, or brain. Combined with the psychoactive properties, this results in feeling "high."19 CBD Results Push Further Research Neurologist Dr. Ethan Russo discussed the difference between THC and CBD and the way they interact with your receptors in an interview with Project CBD. He points out that cannabis has been selectively bred over the years for high THC levels to increase the psychoactive effects of the plant.20 However, the medicinal plant bred for high levels of CBD and low levels of THC do not engage your CBD1 receptors in the same way. He explains: "It doesn't tend to bind directly, what's called the orthosteric site where THC binds. Rather, it binds on what's called an allosteric site, another site on the receptor, and it so it alters the binding of both THC and the endogenous cannabinoids, the endocannabinoids. So, cannabidiol is what's called the negative allosteric modulator, which is a fancy way of saying that when THC is present it interferes with its activity — which is a good thing in terms of wanting too much psychoactivity and again limiting side effects like anxiety or rapid heart rate that can be a problem if someone has too much THC." This difference has fueled a group of "medical cannabis refugees," who have moved to Colorado in search of medicinal plants that have demonstrated unique and powerful success in the treatment of conditions like intractable seizures,21 Parkinson's disease22,23 and obsessive compulsive disorder.24,25 In the past decade the endocannabinoid system has been implicated in a number of biological and physiological functions that show increasing promise in treatment of a wide variety of disorders, including IBD, neuropathic pain and multiple sclerosis.26 However, while great strides have been made in research, the socially unacceptable psychoactive properties of the cannabis plant have presented a significant challenge to families seeking treatment. The use of selective receptor agonists could potentially improve treatment options. How CBD May Affect Your Gut Health Research from the University of Vermont found those who ate red-hot chili peppers experienced a 13 percent reduction in risk of death, primarily from heart disease or stroke.27 Data suggested participants benefited from the result of capsaicin cellular and molecular mechanisms, especially in the gut. The outcome of an excessive inflammatory response in your gut is intestinal damage and disturbances in motility and secretion — the hallmarks of IBD.28 With no known current pharmaceutical cure, success of treatment modalities is measured by severity of symptoms. Active inflammation in the gut may be mediated by enteric glial cells that release factors, amplifying the immune response.29 This represents an important connection between your nervous system and immune system inside your gut. CBD can control these glial cells in your nervous system without psychoactivity, and therefore reduce the inflammatory response in your intestinal tract. These results indicate that CBD meets criteria for a therapeutic strategy to treat IBD.30 The gut-brain connection offers a strong explanation of how your intestinal health responds to stress and, conversely, why acute or chronic abdominal distress may result in emotional complaints. CBD is becoming more recognized for the important role it plays in treating this neuroimmune axis.31 And finally, the side-effect profile for CBD oil and other medicinal preparations of CBD is very low. In fact, the preparations have fewer side effects than current medications used to treat UC and Crohn's disease.32,33 Prevention and Treatment Without Drugs Like most health conditions, prevention is the best medicine. Your gut bacteria have a powerful influence over your immune system and the inflammatory response in your body as I discuss in the video above.34,35 Individuals who suffer from IBD often share an altered microbiome resulting in a protracted state of inflammation and symptoms.36 Reseeding your gut to achieve a more normal composition should be high on your list to reduce your symptoms or prevent the condition. Traditionally fermented and unpasteurized cultured foods are loaded with healthy bacteria, and are easy to make from scratch at home. To learn more about fermenting vegetables, see my previous article, "Tips for Fermenting at Home." Other helpful foods that are important if you struggle with IBD include: • Blueberries. These little berries are packed with antioxidants, vitamins and fiber, and research has demonstrated they may help alleviate and protect against inflammation in your gut.37,38 They are rich in polyphenols that have both antimicrobial and antioxidant effects. When combined with probiotics, blueberries may reduce inflammatory-inducing bacteria in your gut and increase the amount of healthy Lactobacilla. The fiber doesn't degrade in your large intestines, protecting your intestinal lining from substances that cause inflammation. • Coconut oil. This is a wonderful overall addition to your diet and has anti-inflammatory effects that may help heal injury to your digestive tract.39Coconut oil also has antimicrobial properties that may kill microorganisms, including yeast that may cause chronic inflammation. • Caprylic acid. This is a medium-chain triglyceride (MCT oil) that is also a potent antifungal. Dr. Leon Chaitow, author of "Candida Albicans: Could Yeast Be Your Problem?" recommends it in lieu of antifungal drugs for Candida overgrowth. • Animal-based omega-3. This is another essential element for preventing and controlling IBD. The omega-3 fats in krill oil, EPA and DHA, have immune-boosting qualities along with anti-inflammatory properties proven to benefit disorders of the gut, including Crohn's disease and UC.40 • Fiber-rich foods. Fiber foods such as organic psyllium seed husk, flax, hemp and berries, vegetables such as broccoli and Brussels sprouts, root vegetables and tubers, raw nuts and beans, help nourish beneficial microbes in your gut. Aim for 50 grams of fiber per 1,000 calories consumed. Supplementing with organic psyllium husk can help bring you closer to this ideal amount. Just make sure that the psyllium is organic, as non-organic psyllium is typically severely contaminated with pesticides.

[naivelocus]

The Evolution of Lipidomics through Space and Time

Highlights

•

The foundations of mass spectrometry-based lipidomics have evolved over 4 decades.

•

The emergence of lipidomics has been catalyzed by development of new ionization techniques.

•

Advances in resolution and mass accuracy facilitated the growth of the field.

•

Lipidomic data analysis requires consideration of flux and spatial proximity.

•

Reverse lipidomics can identify unknown lipid signaling pathways.

Abstract

Although the foundations of mass spectrometry-based lipidomics have been practiced for over 30 years, recent technological advances in ionization modalities in conjunction with robust increases in mass accuracy and resolution have greatly accelerated the emergence, growth and importance of the field of lipidomics. Moreover, advances in the separation sciences, bioinformatic strategies and the availability of robust databases have been synergistically integrated into modern lipidomic technologies leading to unprecedented improvements in the depth, penetrance and precision of lipidomic analyses and identification of their biological and mechanistic significance. The purpose of this "opinion" article is to briefly review the evolution of lipidomics, critique the platforms that have evolved and identify areas that are likely to emerge in the years to come. Through seamlessly integrating a rich repertoire of mass spectrometric, chemical and bioinformatic strategies, the chemical identities and quantities of tens of thousands to hundreds of thousands of different lipid molecular species and their metabolic alterations during physiologic or pathophysiologic perturbations can be obtained. Thus, the field of lipidomics which already has a distinguished history of exciting new discoveries in many disease states holds unparalleled potential to identify the pleiotropic roles of lipids in health and disease at the chemical level. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Keywords

Lipidomics;

Shotgun lipidomics;

Mass spectrometry;

Reverse lipidomics;

Charge-switch derivatives

1. Introduction

Although the foundations of mass spectrometry-based lipidomics have been practiced for over 30 years, recent technological advances in ionization modalities in conjunction with robust increases in mass accuracy and resolution have greatly accelerated the emergence, growth and importance of the field of lipidomics. Moreover, advances in the separation sciences, bioinformatic strategies and the availability of robust databases have been synergistically integrated into modern lipidomic technologies leading to unprecedented improvements in the depth, penetrance and precision of lipidomic analyses and identification of their biological and mechanistic significance. The purpose of this “opinion” article is to briefly review the evolution of lipidomics, critique the platforms that have evolved and identify areas that are likely to emerge in the years to come. Through seamlessly integrating a rich repertoire of mass spectrometric, chemical and bioinformatic strategies, the chemical identities and quantities of tens of thousands to hundreds of thousands of different lipid molecular species and their metabolic alterations during physiologic or pathophysiologic perturbations can be obtained. Thus, the field of lipidomics which already has a distinguished history of exciting new discoveries in many disease states holds unparalleled potential to identify the pleiotropic roles of lipids in health and disease at the chemical level.

2. The Dawn of Mass Spectrometry-based Lipidomics

Over thirty years ago, we began our adventures in lipidomics exploring the hypothesis that alterations in myocardial membrane lipid constituents result in changes in membrane molecular dynamics and surface charge that precipitate myocardial electrophysiologic dysfunction, hemodynamic failure and sudden death. In the late 1970s, we discovered that negatively charged liposomes and changes in membrane molecular dynamics were potent activators of myocardial phospholipases [1]. However, the methods for identification of alterations in lipid content and composition were severely limited by the difficulties of the laborious techniques used for analysis of the majority of cellular lipids accompanied by corresponding inaccuracies in lipid identification and quantitation. Accordingly, we began the development of lipidomic strategies employing sequential straight and reversed phase HPLC with quantitation by GC-MS to identify alterations in lipid molecular species during cardiac ischemia which had the potential to identify the membrane alterations responsible for lethal ventricular arrhythmias, heart failure and sudden death [2]. At that time, it was clear that a new field needed to be developed which could accurately identify the alterations in cellular membrane constituents to mechanistically understand the roles of lipids in health and disease. In the early 1980s, fast atom bombardment mass spectrometry (FABMS) employing a beam of high-energy Xe atoms focused on a host matrix of glycerol containing peptide guests was developed [3]. We adapted this soft ionization method for lipids resulting in the ionization of the intact phospholipid parent ions with minimal fragmentation. We were astounded when this technology identified plasmalogens (which contain a vinyl ether linkage at the sn-1 position) as the major phospholipid constituents of myocardial sarcolemma [4]. Moreover, ethanolamine glycerophospholipids in sarcolemma were predominantly plasmalogens containing arachidonic acid at the sn-2 position ( Fig. 1) [4]. These results had profound implications for myocardial membrane physical properties and lipid-mediated signal transduction. Subsequent studies using FABMS analysis of sarcoplasmic reticulum and mitochondria demonstrated the abundance of arachidonic acid containing plasmalogens in sarcoplasmic reticulum while mitochondria were comprised predominantly of diacyl phospholipids containing linoleic acid and arachidonic acid [5]. These results demonstrated the profound differences in the lipidome in specific subcellular organelles and their non-uniform spatial distribution in the cell. Critical to the success of these initial lipidomic experiments was the use of HPLC to separate lipid classes followed by soft ionization by FAB to volatize intact phospholipid molecular ions in the absence of significant fragmentation. Collectively, the use of HPLC followed by mass spectrometry identified the class, subclass, molecular species and regiospecificity of ~ 80 phospholipids in canine myocardium many of which were spatially localized in specialized subcellular compartments [4] ;  [5]. The unanticipated predominance of plasmalogens containing almost exclusively arachidonic acid at the sn-2 position in myocardial sarcolemma focused our attention on the chemical, biophysical and signaling properties of plasmalogens and their downstream metabolites. Furthermore, this early technology provided a robust platform for identifying the molecular species of lipids in different cell types and tissues during physiologic or pathophysiologic perturbations. Through the use of these technologies, we identified a diverse array of phospholipid molecular species in human myocardium [6], synaptic vesicles [7] and electrophysiologically active cells [8]. However, the limited sensitivity of FAB ionization prevented its application to large-scale lipidomic analyses.

Fig. 1. 

Plasmalogens Are the Major Storage Depot of Arachidonic Acid in Sarcolemma.Left panel, fast atom bombardment mass spectrometry of sarcolemmal ethanolamine glycerophospholipids performed using highly purified sarcolemma prior to HPLC purification of ethanolamine glycerophospholipids. Approximately 500 pg of ethanolamine glycerophospholipids were dissolved in CHCl3,/CH3OH prior to obtaining fast atom bombardment mass spectra using a Xenon beam to ionize lipids as guests in a glycerol matrix, on a copper probe using a commercially available gas chromatography detector. Right panel, chemical structures of subclasses of ethanolamine diacyl, plasmalogen and alkyl ether glycerophospholipids.

(Biochemistry 1984 Jan3; 23(1):158–165. 0006–2960/84/0423–0158$01.50/0 1984 American Chemical Society.)

Note to users: Accepted manuscripts are Articles in Press that have been peer reviewed and accepted for publication by the Editorial Board of this publication. They have not yet been copy edited and/or formatted in the publication house style, and may not yet have the full ScienceDirect functionality, e.g., supplementary files may still need to be added, links to references may not resolve yet etc. The text could still change before final publication.

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Author : Richard W. Gross — BBA Lipid Molecular and Cell Biology

[rnomics]

Integrative analysis of long noncoding #RNA and #mRNA reveals candidate l#ncRNAs responsible for meat quality at different physiological stages in Gushi chicken

by Donghua Li, Fang Li, Keren Jiang, Meng Zhang, Ruili Han, Ruirui Jiang, Zhuanjian Li, Yadong Tian, Fengbin Yan, Xiangtao Kang, Guirong Sun Long noncoding #RNAs (l#ncRNAs) play important roles in transcriptional and posttranscriptional regulation. However, the effects of l#ncRNAs on the meat quality of chicken hasn’t been elucidated clearly yet. Gushi chickens are popular in China because of their superior meat quality, particularly the tender flesh, and unique flavor. Gushi chickens are popular in China because of their superior meat quality, delicate flesh, and unique flavor. We performed #RNA-Seq analysis of breast muscle from Gushi chicken at two physiological stages, including juvenile (G20W) and laying (G55W). In total, 186 l#ncRNAs and 881 #mRNAs were differentially expressed between G20W and G55W (fold change ≥ 2.0, P < 0.05). Among them, 131 l#ncRNAs presented upregulated and 55 were downregulated. We identified the cis and trans target genes of the differentially expressed l#ncRNAs, and constructed l#ncRNA-#mRNA interaction networks. The results showed that differentially expressed #mRNAs and l#ncRNAs were mainly involved in ECM-receptor interaction, glycerophospholipid metabolism, ubiquitin-mediated proteolysis, and the biosynthesis of amino acids. In summary, our study utilized #RNA-seq analysis to predict the functions of l#ncRNA on chicken meat quality. Furthermore, comprehensive analysis identified l#ncRNAs and their target genes, which may contribute to a better understanding of the molecular mechanisms underlying in poultry meat quality and provide a theoretical basis for further research. http://bit.ly/2WXJYbK