Membrane Fraction Research Articles

The Effect of TGF-β1 Reduced Functionality on the Expression of Selected Synaptic Proteins and Electrophysiological Parameters: Implications of Changes Observed in Acute Hepatic Encephalopathy.

Decreased platelet count represents a feature of acute liver failure (ALF) pathogenesis. Platelets are the reservoir of transforming growth factor 1 (TGF-β1), a multipotent cytokine involved in the maintenance of, i.a., central nervous system homeostasis. Here, we analyzed the effect of a decrease in TGF-β1 active form on synaptic proteins levels, and brain electrophysiology, in mice after intraperitoneal (ip) administration of TGF-β1 antibody (anti-TGF-β1; 1 mg/mL). Next, we correlated it with a thrombocytopenia-induced TGF-β1 decrease, documented in an azoxymethane-induced (AOM; 100 mM ip) model of ALF, and clarified the impact of TGF-β1 decrease on blood–brain barrier functionality. The increase of both synaptophysin and synaptotagmin in the cytosolic fraction, and its reduction in a membrane fraction, were confirmed in the AOM mice brains. Both proteins’ decrease in analyzed fractions occurred in anti-TGF-β1 mice. In turn, an increase in postsynaptic (NR1 subunit of N-methyl-D-aspartate receptor, postsynaptic density protein 95, gephyrin) proteins in the AOM brain cortex, but a selective compensatory increase of NR1 subunit in anti-TGF-β mice, was observed. The alterations of synaptic proteins levels were not translated on electrophysiological parameters in the anti-TGF-β1 model. The results suggest the impairment of synaptic vesicles docking to the postsynaptic membrane in the AOM model. Nevertheless, changes in synaptic protein level in the anti-TGF-β1 mice do not affect neurotransmission and may not contribute to neurologic deficits in AOM mice.

The decreased PG content of pgp1 inhibits PSI photochemistry and limits reaction center and light-harvesting polypeptide accumulation in response to cold acclimation.

Decreased PG constrains PSI activity due to inhibition of transcript and polypeptide abundance of light-harvesting and reaction center polypeptides generating a reversible, yellow phenotype during cold acclimation of pgp1. Cold acclimation of the Arabidopsis pgp1 mutant at 5°C resulted in a pale-yellow phenotype with abnormal chloroplast ultrastructure compared to its green phenotype upon growth at 20°C despite a normal cold-acclimation response at the transcript level. In contrast, wild type maintained its normal green phenotype and chloroplast ultrastructure irrespective of growth temperature. In contrast to cold acclimation of WT, growth of pgp1 at 5°C limited the accumulation of Lhcbs and Lhcas assessed by immunoblotting. However, a novel 43kD polypeptide of Lhcb1 as well as a 29kD polypeptide of Lhcb3 accumulated in the soluble fraction which was absent in the thylakoid membrane fraction of cold-acclimated pgp1 which was not observed in WT. Cold acclimation of pgp1 destabilized the Chl-protein complexes associated with PSI and predisposed energy distribution in favor of PSII rather than PSI compared to the WT. Functionally, in vivo PSI versus PSII photochemistry was inhibited in cold-acclimated pgp1 to a greater extent than in WT relative to controls. Greening of the pale-yellow pgp1 was induced when cold-acclimated pgp1 was shifted from 5 to 20°C which resulted in a marked decrease in excitation pressure to a level comparable to WT. Concomitantly, Lhcbs and Lhcas accumulated with a simultaneous decrease in the novel 43 and 29kD polypeptides. We conclude that the reduced levels of phosphatidyldiacylglycerol in the pgp1 limit the capacity of the mutant to maintain the structure and function of its photosynthetic apparatus during cold acclimation. Thus, maintenance of normal thylakoid phosphatidyldiacylglycerol levels is essential to stabilize the photosynthetic apparatus during cold acclimation.

Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia coli.

Gram-negative diderm bacteria are characterized by a tripartite cell envelope, composed of an inner membrane (IM) and a lipopolysaccharide (LPS)-containing outer membrane (OM), separated by an aqueous spacewherethe peptidoglycan is embedded. LPS is a peculiar glycolipid endowed with several biological activities. The biosynthesis and transport of LPS to its final location take place in every compartment of the cell envelope. Proteins and protein machineries with different subcellular localization are involved in this process to facilitate the trafficking of LPS across subcellular compartments that differ in their physicochemical proprieties. The fractionation of bacterial cell envelopes can give information on the status of the LPS biogenesis by allowing the analysis of LPS profiles and of the localization of proteins involved in the transport. Here, we describe a standardized protocol for membrane fractionation in Escherichia coli using sucrose density gradient centrifugation that separates the IM from the OM cellular fractions. Bacterial cells are first converted into spheroplasts and lysed; then the membrane fractions are collected by ultracentrifugation and separated at high speed by exploiting the differences in membrane density. The fractions obtained are analyzed for LPS total amount and electrophoretic profile.

T1R3 homomeric sweet taste receptor negatively regulates insulin-induced glucose transport through Gαs-mediated microtubules disassembly in 3T3-L1 adipocytes.

T1R3 is a class C G protein-coupled receptor family member that forms heterodimeric umami and sweet taste receptors with T1R1 and T1R2, respectively, in the taste cells of taste buds. T1R3 is expressed in 3T3-L1 cells in homomeric form and negatively regulates adipogenesis in a Gαs-dependent but cAMP-independent manner. Although T1R3 expression is markedly upregulated during adipogenesis, its physiological role in mature adipocytes remains obscure. Here, we show that stimulation of T1R3 with sucralose or saccharin induces microtubule disassembly in differentiated 3T3-L1 adipocytes. The effect was reproduced by treatment with cholera toxin or isoproterenol but not with forskolin. Treatment with sucralose or saccharin for 3 h inhibited insulin-stimulated glucose uptake by 32% and 45% in differentiated adipocytes, respectively, similar to the inhibitory effect of nocodazole (by 33%). Isoproterenol treatment inhibited insulin-stimulated glucose transport by 26%, whereas sucralose did not affect the intrinsic activity of the glucose transporter, indicating that it inhibited insulin-induced GLUT4 translocation to the plasma membrane. Immunostaining analysis showed that insulin-stimulated GLUT4 accumulation on the plasma membrane was abrogated in sucralose-treated cells, in association with depolymerization of microtubules. Sucralose-mediated inhibition of GLUT4 translocation was reversed by the overexpression of dominant-negative Gαs (Gαs-G226A) or knockdown of Gαs. Additionally, membrane fractionation analysis showed that sucralose treatment reduced GLUT4 levels in the plasma membrane fraction from insulin-stimulated adipocytes. We have identified a novel non-gustatory role for homomeric T1R3 in adipocytes, and activation of the T1R3 receptor negatively regulates insulin action of glucose transport via Gαs-dependent microtubule disassembly.

Lipid rafts integrity is essential for prolactin-induced mitogenesis in mouse embryonic stem cells.

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constraints and regulations applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using two-dimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergent-free preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency, we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs act in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

A proteomic approach to identify digestive enzymes, their exocytic and microapocrine secretory routes and their compartmentalization in the midgut of Spodoptera frugiperda.

A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase.

Quantitative Measurement of Extracellular Vesicle Content Delivery Within Acceptor Cells.

Extracellular vesicles (EVs), including exosomes and microvesicles, are thought to transport bioactive molecules from donor to acceptor cells. Although EV uptake has been qualitatively assessed through subcellular imaging, EV content delivery has been rarely addressed due to a lack of adequate methods. Here we present a sensitive bulk assay to quantitatively measure EV uptake and content delivery in mammalian cell. In this assay, EVs containing a NanoLuc luciferase-tagged cargo are mixed with unlabeled acceptor cells. Cell fractionation separates membrane and cytosolic fractions, and luciferase activity is measured within each fraction to determine the percentage of cytosolic release. This assay can be used to further decipher cellular and molecular mechanisms that regulate the EV delivery process or to quantitatively test specific pairs of donor-acceptor cells.

Development of a simple assay to measure glycerophosphodiesterase GDE4 and GDE7 activities for screening their Inhibitors

Lysophosphatidic acid (LPA) is a lipid mediator which regulates various biological processes, including cell proliferation, platelet aggregation, and cancer metastasis. Autotaxin (ATX) is a lysophospholipase D (lyso-PLD)-type enzyme that produces LPA in plasma. In contrast, glycerophosphodiesterase GDE4 and GDE7 (GDEs) are intracellular lyso-PLDs producing LPA. ATX and GDEs may play unique roles, which need to be validated with their potent and selective inhibitors. Although fluorescent substrates are useful for high-throughput screening of enzyme inhibitors, no fluorescent substrates for these GDEs have been reported. Here, we examined whether a fluorescent ATX substrate FS-3 could be applied to such assays. The membrane fractions of GDE4- and GDE7-overexpressing cells hydrolyzed FS-3 in the presence of Mg2+ and Ca2+, respectively. Using these assay systems, we found that several known ATX inhibitors as well as natural substrates lysophosphatidylcholine and lysophosphatidylethanolamine inhibited GDEs. Furthermore, FS-3 was hydrolyzed by the membrane fraction of LNCaP and MCF-7 cells that endogenously express GDE4 and GDE7, respectively. Finally, our assay system could selectively measure GDEs activities in a mixture of the membrane fractions of GDEs-overexpressing cells. These findings allow high-throughput assays of GDEs and lead to the development of selective inhibitors as well as a better understanding of the biological roles of these enzymes.

AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

ABSTRACT The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXФ) (67–70 and 148–151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation. Abbreviations: Amil: amiloride; AP2: adaptor protein complex 2; AP2A1: adaptor related protein complex 2 subunit alpha 1; AP2M1: adaptor related protein complex 2 subunit mu 1; ATG7: autophagy related 7; CAL: calcitriol; Cas9: CRISPR-associated protein 9; Con: control; CPZ: chlorpromazine; DSS: dextran sodium sulfate; EBSS: Earle’s balanced salt solution; IBD: inflammatory bowel disease; TER: trans-epithelial resistance; KD: knockdown; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MβCD: Methyl-β-cyclodextrin; MET: metformin; MG132: carbobenzoxy-Leu-Leu-leucinal; MTOR: mechanistic target of rapamycin kinase; NT: non target; RAPA: rapamycin; RES: resveratrol; SMER: small-molecule enhancer 28; SQSTM1: sequestosome 1; ST: starvation; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Interindividual Variability and Differential Tissue Abundance of Mitochondrial Amidoxime Reducing Component Enzymes in Humans.

Mitochondrial amidoxime-reducing component (mARC) enzymes are molybdenum-containing proteins that metabolize a number of endobiotics and xenobiotics. The interindividual variability and differential tissue abundance of mARC1 and mARC2 were quantified using targeted proteomics in three types of tissue fractions: 1) pediatric liver tissue homogenates, 2) total membrane fraction of the paired liver and kidney samples from pediatric and adult donors, and 3) pooled S9 fractions of the liver, intestine, kidney, lung, and heart. The absolute levels of mARC1 and mARC2 in the pediatric liver homogenate were 40.08 ± 4.26 and 24.58 ± 4.02 pmol/mg homogenate protein, respectively, and were independent of age and sex. In the total membrane fraction of the paired liver and kidney samples, the abundance of hepatic mARC1 and mARC2 was comparable, whereas mARC2 abundance in the kidney was approximately 9-fold higher in comparison with mARC1. The analysis of the third set of samples (i.e., S9 fraction) revealed that mARC1 abundance in the kidney, intestine, and lung was 5- to 13-fold lower than the liver S9 abundance, whereas mARC2 abundance was approximately 3- and 16-fold lower in the intestine and lung than the liver S9, respectively. In contrast, the kidney mARC2 abundance in the S9 fraction was approximately 2.5-fold higher as compared with the hepatic mARC2 abundance. The abundance of mARC enzymes in the heart was below the limit of quantification (∼0.6 pmol/mg protein). The mARC enzyme abundance data presented here can be used to develop physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates. SIGNIFICANCE STATEMENT: A precise targeted quantitative proteomics method was developed and applied to quantify newly discovered drug-metabolizing enzymes, mARC1 and mARC2, in pediatric and adult tissue samples. The data suggest that mARC enzymes are ubiquitously expressed in an isoform-specific manner in the human liver, kidney, intestine, and lung, and the enzyme abundance is not associated with age and sex. These data are important for developing physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.

Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts.

The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.

Metabolism and accumulation of hydroxylated fatty acids by castor (Ricinus comunis) seed microsomes

Castor beans accumulate large amounts of triacylglycerols (TAGs) in the seed endosperm. This oil contains hydroxylated ricinoleic levels close to 90%, which is unique among oil seeds. The capacity to accumulate such high levels of such an unusual fatty acids is due to its specific accumulation and channeling. Here, the ability of the castor biosynthetic machinery to accumulate unusual fatty acids in the form of TAGs was investigated, focusing on ricinoleic acid and the structurally analogous lesquerolic and coriolic fatty acids. The metabolism of different radioactive precursors in active membrane fractions from castor bean's were studied, and the rates and accumulation of these fatty acids provided evidence of the different mechanisms involved in the accumulation of hydroxylated fatty acids in this species. In particular, these studies highlighted the potential of castor to accumulate unusual fatty acids other than ricinoleic acid, showing that castor endosperm can efficiently accumulate lesquerolic acid.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin.

The cellular prion protein (PrPC) is renowned for its infectious conformational isoform PrPSc, capable of templating subsequent conversions of healthy PrPCs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein’s physiological role is also elusive. PrPC and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein–protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.

Regulation of Serotonin 1A Receptor SUMOylation by SENP2 and PIASxα.

Serotonin 1A receptors (5-HT1ARs) are implicated in the control of mood, cognition, and memory and in various neuropsychiatric disorders such as depression and anxiety. As such, understanding the regulation of 5-HT1ARs will inform the development of better treatment approaches. We previously demonstrated 5-HT1ARs are SUMOylated by SUMO1 in the rat brain. Agonist stimulation increased SUMOylation and was further enhanced when combined with 17β-estradiol-3-benzoate (EB), which are treatments that cause the transient and prolonged desensitization of 5-HT1AR signaling, respectively. In the current study, we identified the protein inhibitor of activated STAT (PIAS)xα as the enzyme that facilitates SUMOylation, and SENP2 as the protein that catalyzes the deSUMOylation of 5-HT1ARs. We demonstrated that PIASxα significantly increased in the membrane fraction of rats co-treated with EB and an agonist, compared to either the EB-treated or vehicle-treated groups. The acute treatment with an agonist alone shifted the location of SENP2 from the membrane to the cytoplasmic fraction, but it has little effect on PIASxα. Hence, two separate mechanisms regulate SUMOylation and the activity of 5-HT1ARs by an agonist and EB. The effects of EB on 5-HT1AR SUMOylation and signaling may be related to the higher incidence of mood disorders in women during times with large fluctuations in estrogens. Targeting the SUMOylation of 5-HT1ARs could have important clinical relevance for the therapy for several neuropsychiatric disorders in which 5-HT1ARs are implicated.

Lysosomal subcellular distribution in neurons: Are there synaptic lysosomes?

Despite Alzheimer's disease's first description 114 years ago1 , the mechanisms driving the most common late-onset forms' molecular etiology and neuropathophysiology remain elusive. While considering its elaborate display of pathological hallmarks, recent advances have described synapse loss as the most accurate correlate of disease progression2 . A wide array of data, including insights from human genetics, preclinical AD models, clinical research, and trials, has continuously linked a dysfunctional lysosomal network to pathological aging and disease onset. However, evidence correlating lysosomal dysfunction with early synapse loss is still lacking. Here, we aim to characterize the subcellular distribution of neuronal lysosomes, hypothesizing that these complex organelles localize to both presynaptic and postsynaptic terminals. We have analyzed the subcellular distribution of synaptic and non-synaptic associated LAMP-1+ organelles in our established mature primary hippocampal and cortical mouse neuronal in vitro model and adult mouse brain synaptosomes. We applied quantitative single-cell analysis using ICY and ImageJ software for analytical purposes. Our results show that the subcellular distribution of (endo)lysosomes is highly compartmentalized and dynamic. Based on the % of (endo)lysosomes in the neuronal soma, we documented that roughly 42% of (endo)lysosomes distribute along dendrites and 37% along the axon initial segment (AIS). Remarkably, we report that close to 8% of the total (endo)lysosomal pool was associated with synapsin, a presynaptic compartment marker. We also reveal that about 26% of (endo)lysosomes colocalized with PSD-95, a postsynaptic compartment marker, constituting 60% of the dendritic (endo)lysosomes. Moreover, we found LAMP-1 enriched in mouse brain synaptosomes against nuclear, non-nuclear, non-synaptosomal and membrane fractions. We are currently pushing to characterize these synaptic (endo)lysosomes' function. These results suggest that (endo)lysosomes are present at synapses. In the future we would like to establish whether its dysfunctional activity and trafficking act as a catalyst in the very early stages of synaptic dysfunction in the aging brain, potentially contributing to LOAD etiology and development.

Membrane composition and organization of Bacillus subtilis 168 and its genome-reduced derivative miniBacillus PG10.

SummaryA form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes and offer application possibilities in microbial biotechnology. We designed a lipidomics method to directly measure relative abundances of lipids in detergent‐resistant and detergent‐sensitive membrane fractions of the model bacterium Bacillus subtilis 168 and the biotechnologically attractive miniBacillus PG10 strain. Our study supports previous work suggesting that cardiolipin and prenol lipids are enriched in FMMs of B. subtilis. Additionally, structural analysis of acyl chains of major phospholipids indicated that FMMs display increased order and thickness compared with the surrounding bilayer. Despite the 36% genome reduction, membrane and FMM integrity are largely preserved in miniBacillus PG10, as supported by analysis of membrane fluidity, flotillin distribution and gene expression data. The novel insights in FMM architecture reported here will contribute to further explore the biological significance of FMMs and the means by which FMMs can be exploited as heterologous production platforms. Moreover, our lipidomics method enables comparative FMM lipid profiling between different bacteria.

Stem Cell Mimicking Nanoencapsulation for Targeting Arthritis.

Mesenchymal stem cells (MSCs) are considered a promising regenerative therapy due to their ability to migrate toward damaged tissues. The homing ability of MSCs is unique compared with that of non-migrating cells and MSCs are considered promising therapeutic vectors for targeting major cells in many pathophysiological sites. MSCs have many advantages in the treatment of malignant diseases, particularly rheumatoid arthritis (RA). RA is a representative autoimmune disease that primarily affects joints, and secreted chemokines in the joints are well recognized by MSCs following their migration to the joints. Furthermore, MSCs can regulate the inflammatory process and repair damaged cells in the joints. However, the functionality and migration ability of MSCs injected in vivo still show insufficient. The targeting ability and migration efficiency of MSCs can be enhanced by genetic engineering or modification, eg, overexpressing chemokine receptors or migration-related genes, thus maximizing their therapeutic effect. However, there are concerns about genetic changes due to the increased probability of oncogenesis resulting from genome integration of the viral vector, and thus, clinical application is limited. Furthermore, it is suspected that administering MSCs can promote tumor growth and metastasis in xenograft and orthotopic models. For this reason, MSC mimicking nanoencapsulations are an alternative strategy that does not involve using MSCs or bioengineered MSCs. MSC mimicking nanoencapsulations consist of MSC membrane-coated nanoparticles, MSC-derived exosomes and artificial ectosomes, and MSC membrane-fused liposomes with natural or genetically engineered MSC membranes. MSC mimicking nanoencapsulations not only retain the targeting ability of MSCs but also have many advantages in terms of targeted drug delivery. Specifically, MSC mimicking nanoencapsulations are capable of encapsulating drugs with various components, including chemotherapeutic agents, nucleic acids, and proteins. Furthermore, there are fewer concerns over safety issues on MSC mimicking nanoencapsulations associated with mutagenesis even when using genetically engineered MSCs, because MSC mimicking nanoencapsulations use only the membrane fraction of MSCs. Genetic engineering is a promising route in clinical settings, where nano-encapsulated technology strategies are combined. In this review, the mechanism underlying MSC homing and the advantages of MSC mimicking nanoencapsulations are discussed. In addition, genetic engineering of MSCs and MSC mimicking nanoencapsulation is described as a promising strategy for the treatment of immune-related diseases.

PDGF Promotes Dermal Fibroblast Activation via a Novel Mechanism Mediated by Signaling Through MCHR1.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs. To this day, no effective treatments to prevent the progression of fibrosis exist, and SSc patients have disabilities and reduced life expectancy. The need to better understand pathways that drive SSc and to find therapeutic targets is urgent. RNA sequencing data from SSc dermal fibroblasts suggested that melanin-concentrating hormone receptor 1 (MCHR1), one of the G protein-coupled receptors regulating emotion and energy metabolism, is abnormally deregulated in SSc. Platelet-derived growth factor (PDGF)-BB stimulation upregulated MCHR1 mRNA and protein levels in normal human dermal fibroblasts (NHDF), and MCHR1 silencing prevented the PDGF-BB-induced expression of the profibrotic factors transforming growth factor beta 1 (TGFβ1) and connective tissue growth factor (CTGF). PDGF-BB bound MCHR1 in membrane fractions of NHDF, and the binding was confirmed using surface plasmon resonance (SPR). MCHR1 inhibition blocked PDGF-BB modulation of intracellular cyclic adenosine monophosphate (cAMP). MCHR1 silencing in NHDF reduced PDGF-BB signaling. In summary, MCHR1 promoted the fibrotic response in NHDF through modulation of TGFβ1 and CTGF production, intracellular cAMP levels, and PDGF-BB-induced signaling pathways, suggesting that MCHR1 plays an important role in mediating the response to PDGF-BB and in the pathogenesis of SSc. Inhibition of MCHR1 should be considered as a novel therapeutic strategy in SSc-associated fibrosis.

Characterization of Ictalurid herpesvirus 1 Glycoprotein ORF59 and Its Potential Role on Virus Entry into the Host Cells.

The channel catfish virus (CCV, Ictalurid herpesvirus 1) has caused sustained economic losses in the fish industry because of its strong infectivity and pathogenicity. Thus, it is necessary to determine the function of viral proteins in the CCV infection process. The present study aimed to characterize CCV glycoprotein ORF59 and explore its impact on virus infection in host cells. Firstly, its exclusive presence in the membrane fraction of the cell lysate and subcellular localization verified that CCV ORF59 is a viral membrane protein expressed at late-stage infection. A protein blocking assay using purified His6 tagged ORF59, expressed in sf9 insect cells using a baculovirus expression system, indicated a dose-dependent inhibitory effect of recombinant ORF59 protein on virus invasion. Knockdown of the ORF59 using a short hairpin (shRNA) showed that ORF59 silencing decreased the production of infectious virus particles in channel catfish ovary cells. The results of this study suggest that recombinant ORF59 protein might inhibit CCV entry into the host cells. These findings will promote future studies of the key functions of glycoprotein ORF59 during CCV infection.

The Role of PDE11A4 in Social Isolation-Induced Changes in Intracellular Signaling and Neuroinflammation.

Phosphodiesterase 11A (PDE11A), an enzyme that degrades cyclic nucleotides (cAMP and cGMP), is the only PDE whose mRNA expression in brain is restricted to the hippocampal formation. Previously, we showed that chronic social isolation changes subsequent social behaviors in adult mice by reducing expression of PDE11A4 in the membrane fraction of the ventral hippocampus (VHIPP). Here we seek extend these findings by determining 1) if isolation-induced decreases in PDE11A4 require chronic social isolation or if they occur acutely and are sustained long-term, 2) if isolation-induced decreases occur uniquely in adults (i.e., not adolescents), and 3) how the loss of PDE11 signaling may increase neuroinflammation. Both acute and chronic social isolation decrease PDE11A4 expression in adult but not adolescent mice. This decrease in PDE11A4 is specific to the membrane compartment of the VHIPP, as it occurs neither in the soluble nor nuclear fractions of the VHIPP nor in any compartment of the dorsal HIPP. The effect of social isolation on membrane PDE11A4 is also selective in that PDE2A and PDE10A expression remain unchanged. Isolation-induced decreases in PDE11A4 expression appear to be functional as social isolation elicited changes in PDE11A-relevant signal transduction cascades (i.e., decreased pCamKIIα and pS6-235/236) and behavior (i.e., increased remote long-term memory for social odor recognition). Interestingly, we found that isolation-induced decreases in membrane PDE11A4 correlated with increased expression of interleukin-6 (IL-6) in the soluble fraction, suggesting pro-inflammatory signaling for this cytokine. This effect on IL-6 is consistent with the fact that PDE11A deletion increased microglia activation, although it left astrocytes unchanged. Together, these data suggest that isolation-induced decreases in PDE11A4 may alter subsequent social behavior via increased neuroinflammatory processes in adult mice.