The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation.

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Zulauf, Katelyn E; Sullivan, Jonathan Tabb; Braunstein, Miriam

2018-04-30

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae.

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Chen, Ming-Chyuan; Cheng, Ying-Min; Hong, Min-Chang; Fang, Lee-Shing

2004-11-19

The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts.

Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae

International Nuclear Information System (INIS)

Chen, M.-C.; Cheng, Y.-M; Hong, M.-C.; Fang, L.-S.

2004-01-01

The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts

Autophagosome and phagosome.

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Deretic, Vojo

2008-01-01

Autophagy and phagocytosis are evolutionarily ancient processes functioning in capture and digestion of material found in the cellular interior and exterior, respectively. In their most primordial form, both processes are involved in cellular metabolism and feeding, supplying cells with externally obtained particulate nutrients or using portions of cell's own cytoplasm to generate essential nutrients and energy at times of starvation. Although autophagy and phagocytosis are commonly treated as completely separate biological phenomena, they are topologically similar and can be, at least morphologically, viewed as different manifestations of a spectrum of related processes. Autophagy is the process of sequestering portions of cellular interior (cytosol and intracellular organelles) into a membranous organelle (autophagosome), whereas phagocystosis is its topological equivalent engaged in sequestering cellular exterior. Both autophagosomes and phagosomes mature into acidified, degradative organelles, termed autolysosomes and phagolysosomes, respectively. The basic role of autophagy as a nutritional process, and that of phagocytosis where applicable, has survived in present-day organisms ranging from yeast to man. It has in addition evolved into a variety of specialized processes in metazoans, with a major role in cellular/cytoplasmic homeostasis. In humans, autophagy has been implicated in many health and disease states, including cancer, neurodegeneration, aging and immunity, while phagocytosis plays a role in immunity and tissue homeostasis. Autophagy and phagocytosis cooperate in the latter two processes. In this chapter, we briefly review the regulatory and execution stages of both autophagy and phagocytosis.

Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

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E Wyroba

2009-08-01

Full Text Available Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein and LAMP-2 (lysosomal membrane protein 2 as well as a7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2 in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100 000 x g of equal load were quantified by immunoblotting. LAMP-2 crossreacting polypeptide of ~106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The a7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILPrelated polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells

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Federico Battisti

2017-09-01

Full Text Available Dendritic cells (DCs are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

aprABC: A Mycobacterium tuberculosis complex-specific locus that modulates pH-driven adaptation to the macrophage phagosome

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Abramovitch, Robert B.; Rohde, Kyle H.; Hsu, Fong-Fu; Russell, David G.

2011-01-01

Summary Following phagocytosis by macrophages, Mycobacterium tuberculosis (Mtb) senses the intracellular environment and remodels its gene expression for growth in the phagosome. We have identified an Acid and Phagosome Regulated (aprABC) locus that is unique to the Mtb complex and whose gene expression is induced during growth in acidic environments in vitro and in macrophages. Using the aprA promoter, we generated a strain that exhibits high levels of inducible fluorescence in response to growth in acidic medium in vitro and in macrophages. aprABC expression is dependent on the two-component regulator phoPR, linking phoPR signaling to pH sensing. Deletion of the aprABC locus causes defects in gene expression that impact aggregation, intracellular growth, and the relative levels of storage and cell wall lipids. We propose a model where phoPR senses the acidic pH of the phagosome and induces aprABC expression to fine-tune processes unique for intracellular adaptation of Mtb complex bacteria. PMID:21401735

Coin Tossing Explains the Activity of Opposing Microtubule Motors on Phagosomes.

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Sanghavi, Paulomi; D'Souza, Ashwin; Rai, Ashim; Rai, Arpan; Padinhatheeri, Ranjith; Mallik, Roop

2018-05-07

How the opposing activity of kinesin and dynein motors generates polarized distribution of organelles inside cells is poorly understood and hotly debated [1, 2]. Possible explanations include stochastic mechanical competition [3, 4], coordinated regulation by motor-associated proteins [5-7], mechanical activation of motors [8], and lipid-induced organization [9]. Here, we address this question by using phagocytosed latex beads to generate early phagosomes (EPs) that move bidirectionally along microtubules (MTs) in an in vitro assay [9]. Dynein/kinesin activity on individual EPs is recorded as real-time force generation of the motors against an optical trap. Activity of one class of motors frequently coincides with, or is rapidly followed by opposite motors. This leads to frequent and rapid reversals of EPs in the trap. Remarkably, the choice between dynein and kinesin can be explained by the tossing of a coin. Opposing motors therefore appear to function stochastically and independently of each other, as also confirmed by observing no effect on kinesin function when dynein is inhibited on the EPs. A simple binomial probability calculation based on the geometry of EP-microtubule contact explains the observed activity of dynein and kinesin on phagosomes. This understanding of intracellular transport in terms of a hypothetical coin, if it holds true for other cargoes, provides a conceptual framework to explain the polarized localization of organelles inside cells. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae.

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Chen, Ming-Chyuan; Cheng, Ying-Min; Sung, Ping-Jyun; Kuo, Cham-En; Fang, Lee-Shing

2003-08-29

The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.

Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes

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Brzezinska Agnieszka A

2006-12-01

Full Text Available Abstract Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation.

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Ikeda, Yuka; Kawai, Katsuhisa; Ikawa, Akira; Kawamoto, Kyoko; Egami, Youhei; Araki, Nobukazu

2017-08-01

Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup. © 2017. Published by The Company of Biologists Ltd.

Lysophosphatidylcholine Promotes Phagosome Maturation and Regulates Inflammatory Mediator Production Through the Protein Kinase A–Phosphatidylinositol 3 Kinase–p38 Mitogen-Activated Protein Kinase Signaling Pathway During Mycobacterium tuberculosis Infection in Mouse Macrophages

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Hyo-Ji Lee

2018-04-01

Full Text Available Tuberculosis is caused by the infectious agent Mycobacterium tuberculosis (Mtb. Mtb has various survival strategies, including blockade of phagosome maturation and inhibition of antigen presentation. Lysophosphatidylcholine (LPC is a major phospholipid component of oxidized low-density lipoprotein and is involved in various cellular responses, such as activation of second messengers and bactericidal activity in neutrophils. In this study, macrophages were infected with a low infectious dose of Mtb and treated with LPC to investigate the bactericidal activity of LPC against Mtb. In macrophages infected with Mtb strain, H37Ra or H37Rv, LPC suppressed bacterial growth; however, this effect was suppressed in bone marrow-derived macrophages (BMDMs isolated from G2A (a G protein-coupled receptor involved in some LPC actions knockout mice. LPC also promoted phagosome maturation via phosphatidylinositol 3 kinase (PI3K–p38 mitogen-activated protein kinase (MAPK-mediated reactive oxygen species production and intracellular Ca2+ release during Mtb infection. In addition, LPC induced increased levels of intracellular cyclic adenosine monophosphate (cAMP and phosphorylated glycogen synthase kinase 3 beta (GSK3β in Mtb-infected macrophages. Protein kinase A (PKA-induced phosphorylation of GSK3β suppressed activation of NF-κB in LPC-treated macrophages during Mtb infection, leading to decreased secretion of pro-inflammatory cytokines and increased secretion of anti-inflammatory cytokines. These results suggest that LPC can effectively control Mtb growth by promoting phagosome maturation via cAMP-induced activation of the PKA–PI3K–p38 MAPK pathway. Moreover, LPC can regulate excessive production of pro-inflammatory cytokines associated with bacterial infection of macrophages.

The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

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Michard, Céline; Sperandio, Daniel; Baïlo, Nathalie; Pizarro-Cerdá, Javier; LeClaire, Lawrence; Chadeau-Argaud, Elise; Pombo-Grégoire, Isabel; Hervet, Eva; Vianney, Anne; Gilbert, Christophe; Faure, Mathias; Cossart, Pascale

2015-01-01

ABSTRACT Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. PMID:25944859

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

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Sydor, Tobias; von Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

2013-03-01

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. © 2012 Blackwell Publishing Ltd.

Localization of CORO1A in the Macrophages Containing Mycobacterium leprae

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Suzuki, Koichi; Takeshita, Fumihiko; Nakata, Noboru; Ishii, Norihisa; Makino, Masahiko

2006-01-01

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A

The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

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Min Wang

Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

Chloride flux in phagocytes.

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Wang, Guoshun

2016-09-01

Phagocytes, such as neutrophils and macrophages, engulf microbes into phagosomes and launch chemical attacks to kill and degrade them. Such a critical innate immune function necessitates ion participation. Chloride, the most abundant anion in the human body, is an indispensable constituent of the myeloperoxidase (MPO)-H2 O2 -halide system that produces the potent microbicide hypochlorous acid (HOCl). It also serves as a balancing ion to set membrane potentials, optimize cytosolic and phagosomal pH, and regulate phagosomal enzymatic activities. Deficient supply of this anion to or defective attainment of this anion by phagocytes is linked to innate immune defects. However, how phagocytes acquire chloride from their residing environment especially when they are deployed to epithelium-lined lumens, and how chloride is intracellularly transported to phagosomes remain largely unknown. This review article will provide an overview of chloride protein carriers, potential mechanisms for phagocytic chloride preservation and acquisition, intracellular chloride supply to phagosomes for oxidant production, and methods to measure chloride levels in phagocytes and their phagosomes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

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Youhei Egami

2015-01-01

Full Text Available Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

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Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

2008-12-22

Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification

Mycobacteria, Metals, and the Macrophage

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Niederweis, Michael; Wolschendorf, Frank; Mitra, Avishek; Neyrolles, Olivier

2015-01-01

Summary Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies. PMID:25703564

Rab39a interacts with phosphatidylinositol 3-kinase and negatively regulates autophagy induced by lipopolysaccharide stimulation in macrophages.

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Shintaro Seto

Full Text Available Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K. Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34(th to 41(st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.

Cryptococcus neoformans: Tripping on Acid in the phagolysosome

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Carlos Miguel De Leon Rodriguez

2016-02-01

Full Text Available Cryptococcus neoformans (Cn is a basidiomycetous pathogenic yeast that is a frequent cause of meningoencephalitis in immunocompromised individuals. Cn is a facultative intracellular pathogen in mammals, insects and amoeba. Cn infection occurs after inhalation of spores or desiccated cells from the environment. After inhalation Cn localizes to the lungs where it can be phagocytosed by alveolar macrophages. Cn is surrounded by a polysaccharide capsule that helps the fungus survive in vivo by interfering with phagocytosis, quenching free radical bursts and shedding polysaccharides that negatively modulates the immune system. After phagocytosis, Cn resides within the phagosome that matures to become a phagolysosome, a process that results in the acidification of the phagolysosomal lumen. Cn replicates at a higher rate inside macrophages than in the extracellular environment, possibly as a result that the phagosomal pH is near that optimal for growth. Cn increases the phagolysosomal pH and modulates the dynamics of Rab GTPases interaction with the phagolysosome. Chemical manipulation of the phagolysosomal pH with drugs can result in direct and indirect killing of Cn and reduced non-lytic exocytosis. Phagolysosomal membrane damage after Cn infection occurs both in vivo and in vitro, and is required for Cn growth and survival. Macrophage treatment with IFN-γ reduces the phagolysosomal damage and increases intracellular killing of Cn. Studies on mice and humans show that treatment with IFN-γ can improve host control of the disease. However the mechanism by which Cn mediates phagolysosomal membrane damage remains unknown but likely candidates are phospholipases and mechanical damage from an enlarging capsule. Here we review Cn intracellular interaction with a particular emphasis on phagosomal interactions and develop the notion that the extent of damage of the phagosomal membrane is a key determinant of the outcome of the Cn-macrophage interaction.

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages.

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Toma, Claudia; Okura, Nobuhiko; Takayama, Chitoshi; Suzuki, Toshihiko

2011-11-01

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non-pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow-derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans-containing phagosomes lagged behind that of L. biflexa-containing phagosomes, and although bone marrow-derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane-bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs. © 2011 Blackwell Publishing Ltd.

Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

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Ohlsson, Lars; Exley, Christopher; Darabi, Anna; Sandén, Emma; Siesjö, Peter; Eriksson, Håkan

2013-11-01

Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles. © 2013.

The role of lipids in host microbe interactions.

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Lang, Roland; Mattner, Jochen

2017-06-01

Lipids are one of the major subcellular constituents and serve as signal molecules, energy sources, metabolic precursors and structural membrane components in various organisms. The function of lipids can be modified by multiple biochemical processes such as (de-)phosphorylation or (de-)glycosylation, and the organization of fatty acids into distinct cellular pools and subcellular compartments plays a pivotal role for the morphology and function of various cell populations. Thus, lipids regulate, for example, phagosome formation and maturation within host cells and thus, are critical for the elimination of microbial pathogens. Vice versa, microbial pathogens can manipulate the lipid composition of phagosomal membranes in host cells, and thus avoid their delivery to phagolysosomes. Lipids of microbial origin belong also to the strongest and most versatile inducers of mammalian immune responses upon engagement of distinct receptors on myeloid and lymphoid cells. Furthermore, microbial lipid toxins can induce membrane injuries and cell death. Thus, we will review here selected examples for mutual host-microbe interactions within the broad and divergent universe of lipids in microbial defense, tissue injury and immune evasion.

Macrophage functions measured by magnetic microparticles in vivo and in vitro

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Moeller, Winfried; Kreyling, Wolfgang G.; Kohlhaeufl, Martin; Haeussinger, Karl; Heyder, Joachim

2001-01-01

Monodisperse ferrimagnetic iron-oxide particles of 1.4 μm geometric diameter were used to study alveolar macrophage functions (phagocytosis, phagosome transport) and cytoskeletal integrity in healthy subjects and in patients with idiopathic pulmonary fibrosis as well as in cultured macrophages. Dysfunctions in phagocytosis, in phagosome transport and cytoskeletal integrity correlated with an impaired alveolar clearance and could be induced in vitro by cytoskeletal drugs

Macrophage functions measured by magnetic microparticles in vivo and in vitro

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Moeller, Winfried E-mail: moeller@gsf.de; Kreyling, Wolfgang G.; Kohlhaeufl, Martin; Haeussinger, Karl; Heyder, Joachim

2001-07-01

Monodisperse ferrimagnetic iron-oxide particles of 1.4 {mu}m geometric diameter were used to study alveolar macrophage functions (phagocytosis, phagosome transport) and cytoskeletal integrity in healthy subjects and in patients with idiopathic pulmonary fibrosis as well as in cultured macrophages. Dysfunctions in phagocytosis, in phagosome transport and cytoskeletal integrity correlated with an impaired alveolar clearance and could be induced in vitro by cytoskeletal drugs.

Rapamycin-based inducible translocation systems for studying phagocytosis.

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Bohdanowicz, Michal; Fairn, Gregory D

2011-01-01

Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent -phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetically encoded rapamycin--induced heterodimerization system. In the presence of rapamycin, tight association between FK506-binding protein (FKBP) and FKBP rapamycin-binding protein (FRB) is observed. Based on this principle, a synthetic system consisting of a targeting domain attached to FKBP can recruit a protein of interest fused to FRB upon the addition of rapamycin. Previously, this technique has been used to target lipid-modifying enzymes and small GTPases to the phagosome or plasma membrane. The recruitment of the FRB module can be monitored by fluorescent microscopy if a fluorescent protein is fused to the FRB sequence. While the focus of this chapter is on phagocytic events, this method can be employed to study any organelle of interest when the appropriate targeting sequence is used.

Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

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Alexandre Gouzy

2014-02-01

Full Text Available Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s, that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

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Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

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Venkateswara Reddy Gogulamudi

2015-10-01

Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

Stage-specific sampling by pattern recognition receptors during Candida albicans phagocytosis.

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Sigrid E M Heinsbroek

2008-11-01

Full Text Available Candida albicans is a medically important pathogen, and recognition by innate immune cells is critical for its clearance. Although a number of pattern recognition receptors have been shown to be involved in recognition and phagocytosis of this fungus, the relative role of these receptors has not been formally examined. In this paper, we have investigated the contribution of the mannose receptor, Dectin-1, and complement receptor 3; and we have demonstrated that Dectin-1 is the main non-opsonic receptor involved in fungal uptake. However, both Dectin-1 and complement receptor 3 were found to accumulate at the site of uptake, while mannose receptor accumulated on C. albicans phagosomes at later stages. These results suggest a potential role for MR in phagosome sampling; and, accordingly, MR deficiency led to a reduction in TNF-alpha and MCP-1 production in response to C. albicans uptake. Our data suggest that pattern recognition receptors sample the fungal phagosome in a sequential fashion.

Burkholderia pseudomallei Evades Nramp1 (Slc11a1- and NADPH Oxidase-Mediated Killing in Macrophages and Exhibits Nramp1-Dependent Virulence Gene Expression

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Veerachat Muangsombut

2017-08-01

Full Text Available Bacterial survival in macrophages can be affected by the natural resistance-associated macrophage protein 1 (Nramp1; also known as solute carrier family 11 member a1 or Slc11a1 which localizes to phagosome membranes and transports divalent cations, including iron. Little is known about the role of Nramp1 in Burkholderia infection, in particular whether this differs for pathogenic species like Burkholderia pseudomallei causing melioidosis or non-pathogenic species like Burkholderia thailandensis. Here we show that transfected macrophages stably expressing wild-type Nramp1 (Nramp1+ control the net replication of B. thailandensis, but not B. pseudomallei. Control of B. thailandensis was associated with increased cytokine responses, and could be abrogated by blocking NADPH oxidase-mediated production of reactive oxygen species but not by blocking generation of reactive nitrogen species. The inability of Nramp1+ macrophages to control B. pseudomallei was associated with rapid escape of bacteria from phagosomes, as indicated by decreased co-localization with LAMP1 compared to B. thailandensis. A B. pseudomallei bipB mutant impaired in escape from phagosomes was controlled to a greater extent than the parent strain in Nramp1+ macrophages, but was also attenuated in Nramp1− cells. Consistent with reduced escape from phagosomes, B. thailandensis formed fewer multinucleated giant cells in Nramp1+ macrophages at later time points compared to B. pseudomallei. B. pseudomallei exhibited elevated transcription of virulence-associated genes of Type VI Secretion System cluster 1 (T6SS-1, the Bsa Type III Secretion System (T3SS-3 and the bimA gene required for actin-based motility in Nramp1+ macrophages. Nramp1+ macrophages were found to contain decreased iron levels that may impact on expression of such genes. Our data show that B. pseudomallei is able to evade Nramp1- and NADPH oxidase-mediated killing in macrophages and that expression of virulence

Restoration of growth by manganese in a mutant strain of Escherichia coli lacking most known iron and manganese uptake systems

DEFF Research Database (Denmark)

Taudte, Nadine; German, Nadezhda; Zhu, Yong-Guan

2016-01-01

The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron/manganese-uptake ......The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron...

Salt, chloride, bleach, and innate host defense

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Wang, Guoshun; Nauseef, William M.

2015-01-01

Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

Salt, chloride, bleach, and innate host defense.

Science.gov (United States)

Wang, Guoshun; Nauseef, William M

2015-08-01

Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. © Society for Leukocyte Biology.

Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

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Michael G Connor

2015-10-01

Full Text Available Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

Purification and proteomics of pathogen-modified vacuoles and membranes

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Jo-Ana eHerweg

2015-06-01

Full Text Available Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e. the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

The Recombinant Bacille Calmette–Guérin Vaccine VPM1002: Ready for Clinical Efficacy Testing

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Natalie E. Nieuwenhuizen

2017-09-01

Full Text Available The only licensed vaccine against tuberculosis (TB, bacille Calmette–Guérin (BCG, protects against severe extrapulmonary forms of TB but is virtually ineffective against the most prevalent form of the disease, pulmonary TB. BCG was genetically modified at the Max Planck Institute for Infection Biology to improve its immunogenicity by replacing the urease C encoding gene with the listeriolysin encoding gene from Listeria monocytogenes. Listeriolysin perturbates the phagosomal membrane at acidic pH. Urease C is involved in neutralization of the phagosome harboring BCG. Its depletion allows for rapid phagosome acidification and promotes phagolysosome fusion. As a result, BCGΔureC::hly (VPM1002 promotes apoptosis and autophagy and facilitates release of mycobacterial antigens into the cytosol. In preclinical studies, VPM1002 has been far more efficacious and safer than BCG. The vaccine was licensed to Vakzine Projekt Management and later sublicensed to the Serum Institute of India Pvt. Ltd., the largest vaccine producer in the world. The vaccine has passed phase I clinical trials in Germany and South Africa, demonstrating its safety and immunogenicity in young adults. It was also successfully tested in a phase IIa randomized clinical trial in healthy South African newborns and is currently undergoing a phase IIb study in HIV exposed and unexposed newborns. A phase II/III clinical trial will commence in India in 2017 to assess efficacy against recurrence of TB. The target indications for VPM1002 are newborn immunization to prevent TB as well as post-exposure immunization in adults to prevent TB recurrence. In addition, a Phase I trial in non-muscle invasive bladder cancer patients has been completed, and phase II trials are ongoing. This review describes the development of VPM1002 from the drawing board to its clinical assessment.

Intracellular events regulating cross-presentation

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Peter eCresswell

2012-06-01

Full Text Available Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation (ERAD machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on MHC class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation.

Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

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Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

2014-01-01

Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

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Burgoyne, Thomas; Lane, Amelia; Laughlin, William E.; Cheetham, Michael E.

2018-01-01

Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM. PMID:29315318

Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway.

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Kim, Tae Sung; Shin, Yern-Hyerk; Lee, Hye-Mi; Kim, Jin Kyung; Choe, Jin Ho; Jang, Ji-Chan; Um, Soohyun; Jin, Hyo Sun; Komatsu, Masaaki; Cha, Guang-Ho; Chae, Han-Jung; Oh, Dong-Chan; Jo, Eun-Kyeong

2017-06-13

The induction of host cell autophagy by various autophagy inducers contributes to the antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain that causes human tuberculosis. In this study, we present a role for the newly identified cyclic peptides ohmyungsamycins (OMS) A and B in the antimicrobial responses against Mtb infections by activating autophagy in murine bone marrow-derived macrophages (BMDMs). OMS robustly activated autophagy, which was essentially required for the colocalization of LC3 autophagosomes with bacterial phagosomes and antimicrobial responses against Mtb in BMDMs. Using a Drosophila melanogaster-Mycobacterium marinum infection model, we showed that OMS-A-induced autophagy contributed to the increased survival of infected flies and the limitation of bacterial load. We further showed that OMS triggered AMP-activated protein kinase (AMPK) activation, which was required for OMS-mediated phagosome maturation and antimicrobial responses against Mtb. Moreover, treating BMDMs with OMS led to dose-dependent inhibition of macrophage inflammatory responses, which was also dependent on AMPK activation. Collectively, these data show that OMS is a promising candidate for new anti-mycobacterial therapeutics by activating antibacterial autophagy via AMPK-dependent signaling and suppressing excessive inflammation during Mtb infections.

A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

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Brown, Roslyn N.; Sanford, James A.; Park, Jea H.; Deatherage, Brooke L.; Champion, Boyd L.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

2012-06-01

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and infection-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of over 30% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB, PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein location for Salmonella and a framework for further investigations using computational modeling.

Regulation of Three Virulence Strategies of Mycobacterium tuberculosis: A Success Story

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van Dam, Jesse C. J.; Martins dos Santos, Vitor A. P.

2018-01-01

Tuberculosis remains one of the deadliest diseases. Emergence of drug-resistant and multidrug-resistant M. tuberculosis strains makes treating tuberculosis increasingly challenging. In order to develop novel intervention strategies, detailed understanding of the molecular mechanisms behind the success of this pathogen is required. Here, we review recent literature to provide a systems level overview of the molecular and cellular components involved in divalent metal homeostasis and their role in regulating the three main virulence strategies of M. tuberculosis: immune modulation, dormancy and phagosomal rupture. We provide a visual and modular overview of these components and their regulation. Our analysis identified a single regulatory cascade for these three virulence strategies that respond to limited availability of divalent metals in the phagosome. PMID:29364195

DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity.

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Jeanette E Bröms

Full Text Available The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI, which is believed to encode a type VI secretion system (T6SS. In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both ΔvgrG and ΔdotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis.

Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D

DEFF Research Database (Denmark)

Holmskov, U; Mollenhauer, J; Madsen, J

1999-01-01

in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2, 413 amino acids. The domain organization features 13 scavenger receptor cysteine...... in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small...

Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

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María Milagros López de Armentia

2016-03-01

Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

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Douglas R Boettner

2008-01-01

Full Text Available Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK, was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i incubation of ameba with anti-PATMK antibodies; (ii PATMK mRNA knock-down using a novel shRNA expression system; and (iii expression of a carboxy-truncation of PATMK (PATMK(delta932. Expression of the carboxy-truncation of PATMK(delta932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

Science.gov (United States)

Boettner, Douglas R; Huston, Christopher D; Linford, Alicia S; Buss, Sarah N; Houpt, Eric; Sherman, Nicholas E; Petri, William A

2008-01-01

Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

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Kristine Porter

Full Text Available Cells in the trabecular meshwork (TM, a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment. Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB. Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light

International Nuclear Information System (INIS)

Besharse, J.C.; Hollyfield, J.G.; Rayborn, M.E.

1977-01-01

The rate of disk addition to rod outer segments (ROS) varies widely in Xenopus laevis tadpoles kept in cyclic light (12L:12D). When measured as radioactive band ( 3 H-band) displacement during the 2nd day after injection of [ 3 H]leucine, 75% of the daily increment of displacement occurred during the first 8 h of light. During the same interval, the number of open disks at the ROS base increased more than threefold. During the last 8 h of darkness, 3 H-band displacement was undetectable and the number of open disks was reduced. These observations suggest the possibility that disk addition may occur discontinuously. During the 3rd and 4th days after injection of [ 3 H]leucine, maximal displacement of the 3 H-band occurred later in the day than on the 2nd day, its movement no longer corresponding to the increase in open disks. This delay in 3 H-band displacement may reflect a time delay as a result of propagation of compressive stress in an elastic ROS system. Maximal disk loss from ROS as reflected in counts of phagosomes in the pigment epithelium occurred within 1 h of light exposure, and phagosome counts remained high for 4 h before declining to a low level in darkness. Modified lighting regimes affected the daily rhythms of shedding and disk addition differently, suggesting that control mechanisms for the two processes are not directly coupled. During 3 days in darkness, disk addition was reduced 50% compared to controls (12L:12D), whereas shedding was reduced by about 40%. Although reduced in level, shedding occurred as a free-running circadian rhythm

Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium

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Brown David M

2005-10-01

Full Text Available Abstract Background Particulate air pollution is reported to cause adverse health effects in susceptible individuals. Since most of these particles are derived form combustion processes, the primary composition product is carbon with a very small diameter (ultrafine, less than 100 nm in diameter. Besides the induction of reactive oxygen species and inflammation, ultrafine particles (UFP can cause intracellular calcium transients and suppression of defense mechanisms of alveolar macrophages, such as impaired migration or phagocytosis. Methods In this study the role of intracellular calcium transients caused by UFP was studied on cytoskeleton related functions in J774A.1 macrophages. Different types of fine and ultrafine carbon black particles (CB and ufCB, respectively, such as elemental carbon (EC90, commercial carbon (Printex 90, diesel particulate matter (DEP and urban dust (UD, were investigated. Phagosome transport mechanisms and mechanical cytoskeletal integrity were studied by cytomagnetometry and cell viability was studied by fluorescence microscopy. Macrophages were exposed in vitro with 100 and 320 μg UFP/ml/million cells for 4 hours in serum free medium. Calcium antagonists Verapamil, BAPTA-AM and W-7 were used to block calcium channels in the membrane, to chelate intracellular calcium or to inhibit the calmodulin signaling pathways, respectively. Results Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 μg/ml/million cells and above, but not with DEP or UD. Verapamil and W-7, but not BAPTA-AM inhibited the cytoskeletal dysfunctions caused by EC90 or P90. Additionally the presence of 5% serum or 1% bovine serum albumin (BSA suppressed the cytoskeletal dysfunctions. Cell viability showed similar results, where co-culture of ufCB together with Verapamil, W-7, FCS or BSA produced less cell dead compared to the particles only.

A method for functional trans-complementation of intracellular Francisella tularensis.

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Shaun Steele

Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

Fluorescence microscopic and microautoradiographic studies on apoptosis of bone tumor cells induced by 153Sm-EDTMP

International Nuclear Information System (INIS)

Zhu Shoupeng; Xiao Dong; Han Xiaofeng

1997-09-01

The apoptosis of bone tumor cells treated with internal irradiation by 153 Sm-EDTMP was studied. The morphological changes in bone tumor cells were observed by fluorescence microscopic and microautoradiographic observations. It was found that bone tumor cells internally irradiated with 153 Sm-EDTMP, displayed significant nuclear fragmentation and marked pyknosis as well as apoptotic bodies formation. The microautoradiographic study showed that 153 Sm-EDTMP could permeate through cell membrane and displayed membrane-seeking condensation in tumor cells. Soon afterwards 153 Sm-EDTMP could be phagocytized by the tumor cells and distributed in cytoplasm and nucleus in the form of phagosome. With the prolongation of observing time, the membrane-bounded apoptotic bodies was observed. With the lengthening of internal irradiation time by 153 Sm-EDTMP, the inhibition rate of proliferation of bone tumor cells increased progressively. (10 refs., 9 figs., 1 tab.)

Mallik, Prof. Roop

Indian Academy of Sciences (India)

Specialization: Molecular Motors, Phagosome Dynamics, Lipid Droplets, Triglyceride Secretion from Liver Address: Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, Maharashtra Contact: Office: (022) 2278 2702. Residence: (022) 2280 4703. Fax: (022) ...

Vitamin E--a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes

International Nuclear Information System (INIS)

Butterick, C.J.; Baehner, R.L.; Boxer, L.A.; Jersild, R.A. Jr.

1983-01-01

The cellular sites of H 2 O 2 formation in phagocytizing granulocytes have been identified with cerium chloride. A precipitate was visible in phagosomes and on plasma membranes from intact normal cells in the presence of either 0.71 mM NADH or NADPH. X-ray microanalysis permitted identification of cerium deposition within the phagosomes even in the absence of reduced pyridine nucleotides. Catalase ablated the formation of the reaction product. Intact granulocytes obtained from subjects receiving 1600 units of vitamin E daily for 2 weeks exhibited reaction product in the presence of NADH but not NADPH. Intact cells from subjects treated with vitamin E demonstrated diminished numbers of phagocytic vesicles containing reaction product. During phagocytosis the granulocytes treated with vitamin E consumed oxygen but exhibited significantly reduced rates of hydrogen-peroxide-dependent glucose-1- 14 C oxidation to 14 CO 2 . Isolated phagocytic vesicles obtained from granulocytes after ingestion of opsonized lipopolysaccharide-paraffin oil droplets contained reaction product when exposed to 0.71 mM NADPH. No reaction product was evident at 0.71 mM NADH but was evident at 2.0 mM NADH. Isolated phagocytic vesicles from the granulocytes of subjects receiving vitamin E exhibited reaction product only in the presence of NADH. These observations suggest that vitamin E interferes with the electron transport chain apparently required for the oxidation of NADPH to form H 2 O 2 in the phagocytizing granulocyte

Palmitoylation of the immunity related GTPase, Irgm1: impact on membrane localization and ability to promote mitochondrial fission.

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Stanley C Henry

Full Text Available The Immunity-Related GTPases (IRG are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.

Efferocytosis is impaired in Gaucher macrophages.

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Aflaki, Elma; Borger, Daniel K; Grey, Richard J; Kirby, Martha; Anderson, Stacie; Lopez, Grisel; Sidransky, Ellen

2017-04-01

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramide-laden macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67 phox and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67 phox to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease. Copyright© Ferrata Storti Foundation.

Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

DEFF Research Database (Denmark)

Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

2006-01-01

A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG.

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Wang, Jinli; Huang, Chunyu; Wu, Minhao; Zhong, Qiu; Yang, Kun; Li, Miao; Zhan, Xiaoxia; Wen, Jinsheng; Zhou, Lin; Huang, Xi

2015-04-01

To explore the role of myeloid-related protein 8/14 in mycobacterial infection. The mRNA and protein expression levels of MRP8 or MRP14 were measured by real-time PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry. MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROS-dependent manner. The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

THE DIFFERENCE OF MAP1LC3 LEVEL AS MACROPHAGE AUTOPHAGY MARKER BETWEEN RESISTANT AND SENSITIVE TUBERCULOSIS PATIENTS ON RIFAMPICIN

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Dian novita W

2018-04-01

Full Text Available Mycobacterium tuberculosis (MTB is an intracelular bacteria that live in the host macrophage cells. Several organs can be affected by tuberculosis but most major illnesses are lung diseases. Immediately after infection, MTB will be phagocytosed by the alveolar macrophage cells and can survive in the phagosome. The macrophage plays a role in innate immunity towards an infection using autophagy by removing the microbe directly via phagocytosis. When bacteria phagocytosized, vacuole membrane formed double membranes called autophagosome, and followed by degradation by lysosome, which known as autolysosome. Induction of autophagy can be observed on the formation of microtubule-associated proteins 1B lightchain 3B (MAP1LC3B/LC3. MAP1LC3B is protein that have role at autophagic way for selection autophagy substrate and biogenesis. In this study we are used serum from patients TB with rifampicin resistant and rifampicin sensitive as control. Samples were divided using gene expert to differentiate between resistant and sensitive rifampicin.This research aims to compare MAP1LC3B levels in resistant and sensitive rifampicin to study macrophages respond in autophagic way in tuberculosis patients, and give information for define therapy plan to improve therapy for MDR-TB patients. Type of this research is a case control study design with cross sectional research with each groups sample is 19 from age 18-65 years old. Result, MAP1LC3B serum levels on the rifampicin resistant group are lower compared to rifampicin sensitive group. This occur because MTB is able to hide and evade innate immune defense mechanisms. MTB can maintain intracellular growth inside the phagosome by inhibiting phagolysosome formation in autophagy process especially inhibit MAP1LC3B formation by PDIM.

Chronic Granulomatous Disease: The European Experience

NARCIS (Netherlands)

van den Berg, J. Merlijn; van Koppen, Elsbeth; Ahlin, Anders; Belohradsky, Bernd H.; Bernatowska, Ewa; Corbeel, Lucien; Español, Teresa; Fischer, Alain; Kurenko-Deptuch, Magdalena; Mouy, Richard; Petropoulou, Theoni; Roesler, Joachim; Seger, Reinhard; Stasia, Marie-José; Valerius, Niels H.; Weening, Ron S.; Wolach, Baruch; Roos, Dirk; Kuijpers, Taco W.

2009-01-01

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst'', essential for the clearance of phagocytosed

Chronic granulomatous disease: the European experience

NARCIS (Netherlands)

van den Berg, J.M.; van Koppen, E.; Åhlin, A.; Belohradsky, B.H.; Bernatowska, E.; Corbeel, L.; Español, T.; Fischer, A.; Kurenko-Deptuch, M.; Mouy, R.; Petropoulou, T.; Roesler, J.; Seger, R.; Stasia, M.J.; Valerius, N.H.; Weening, R.S.; Wolach, B.; Roos, D.; Kuijpers, T.W.

2009-01-01

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst'', essential for the clearance of phagocytosed

Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

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Elaine F Kenny

Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection.

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Ladomersky, Erik; Khan, Aslam; Shanbhag, Vinit; Cavet, Jennifer S; Chan, Jefferson; Weisman, Gary A; Petris, Michael J

2017-09-01

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a LysMcre mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection. Copyright © 2017 American Society for Microbiology.

Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms.

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Hwang, Sejung; Bang, Kyeongrin; Lee, Jiae; Cho, Saeyoull

2015-01-01

Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing.

Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus (Coleoptera: Scarabaeidae and the Cellular Immune Response to Microorganisms.

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Sejung Hwang

Full Text Available Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus (Coleoptera: Scarabaeidae were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36% compared to controls (5.08%, as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3 to the phagosome, which may result in enhanced pathogen killing.

Phospholipase A and the interaction of Rickettsia prowazekii and mouse fibroblasts (L-929 cells)

International Nuclear Information System (INIS)

Winkler, H.H.; Miller, E.T.

1982-01-01

L-929 cells were killed when approximately 50 viable Rickettsia prowazekii organisms per L-cell were centrifuged onto a monolayer. The glycerophospholipids of the L-cell were hydrolyzed to lysophosphatides and free fatty acids. Concomitantly, there was a loss of membrane integrity as shown by release of lactate dehydrogenase and 86Rb and permeability to trypan blue dye. No glycerophospholipid hydrolysis or cytotoxicity occurred when the rickettsiae were inactivated by heat, UV irradiation, N-ethylmaleimide, or metabolic inhibitors before their addition to the L-929 cells. On the other hand, treatment of the L929 cells with the cytoskeleton agents colchicine or cytochalasin B or with N-ethylmaleimide inhibited neither the phospholipase A activity nor the loss of membrane integrity. Cytochalasin B-treated cells could be damaged by even small numbers of rickettsiae. We suggest that this phospholipase A activity is used by the rickettsiae to escape from the phagosomes into the cytoplasm of host cells

The molecular biology and diagnostics of Chlamydia trachomatis

DEFF Research Database (Denmark)

Birkelund, Svend

1992-01-01

The rapid development of biotechnological methods provides the potential of dissecting the molecular structure of microorganisms. In this review the molecular biology of chlamydia is described. The genus Chlamydia contains three species C. trachomatis, C. psittaci, and C. pneumonia which all...... are important human pathogens. Chlamydia is obligate intracellular bacteria with a unique biphasic life cycle. The extracellularly chlamydial elementary bodies (EB) are small, metabolic inactive, infectious particles with a tight outer cell membrane. After internalization into host cells the chlamydial...... of chlamydia have not yet been found. The adhesin(s) is unknown, and no factor of importance for the inhibition of fusion between phagosome and host cell lysosomes has been described. A protein similar to the mip gene product of Legionella pneumofila may be a possible candidate for a pathogenicity factor...

TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation

NARCIS (Netherlands)

Nair-Gupta, Priyanka; Baccarini, Alessia; Tung, Navpreet; Seyffer, Fabian; Florey, Oliver; Huang, Yunjie; Banerjee, Meenakshi; Overholtzer, Michael; Roche, Paul A.; Tampé, Robert; Brown, Brian D.; Amsen, Derk; Whiteheart, Sidney W.; Blander, J. Magarian

2014-01-01

Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly

Immune evasion by a staphylococcal inhibitor of myeloperoxidase

Science.gov (United States)

de Jong, Nienke W. M.; Ramyar, Kasra X.; Guerra, Fermin E.; Fevre, Cindy; Voyich, Jovanka M.; McCarthy, Alex J.; Garcia, Brandon L.; van Kessel, Kok P. M.; van Strijp, Jos A. G.; Geisbrecht, Brian V.; Haas, Pieter-Jan A.

2017-01-01

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing. PMID:28808028

Immune evasion by a staphylococcal inhibitor of myeloperoxidase

Energy Technology Data Exchange (ETDEWEB)

de Jong, Nienke W. M.; Ramyar, Kasra X.; Guerra, Fermin E.; Nijland, Reindert; Fevre, Cindy; Voyich, Jovanka M.; McCarthy, Alex J.; Garcia, Brandon L.; van Kessel, Kok P. M.; van Strijp, Jos A. G.; Geisbrecht, Brian V.; Haas, Pieter-Jan A.

2017-08-14

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein “staphylococcal peroxidase inhibitor” (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

Vitamin D Is Required for IFN-γ–Mediated Antimicrobial Activity of Human Macrophages

Science.gov (United States)

Fabri, Mario; Stenger, Steffen; Shin, Dong-Min; Yuk, Jae-Min; Liu, Philip T.; Realegeno, Susan; Lee, Hye-Mi; Krutzik, Stephan R.; Schenk, Mirjam; Sieling, Peter A.; Teles, Rosane; Montoya, Dennis; Iyer, Shankar S.; Bruns, Heiko; Lewinsohn, David M.; Hollis, Bruce W.; Hewison, Martin; Adams, John S.; Steinmeyer, Andreas; Zügel, Ulrich; Cheng, Genhong; Jo, Eun-Kyeong; Bloom, Barry R.; Modlin, Robert L.

2012-01-01

Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D–dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D–sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D–deficient serum with 25-hydroxyvitamin D3 restored IFN-γ–induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection. PMID:21998409

Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis

Science.gov (United States)

Lauer, Sabine A.; Iyer, Srinivas; Sanchez, Timothy; Forst, Christian V.; Bowden, Brent; Carlson, Kay; Sriranganathan, Nammalwar; Boyle, Stephen M.

2014-01-01

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide

Intracellular invasion of Orientia tsutsugamushi activates inflammasome in asc-dependent manner.

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Jung-Eun Koo

Full Text Available Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium, which escapes from the endo/phagosome and replicates in the host cytoplasm. O. tsutsugamushi infection induces production of pro-inflammatory mediators including interleukin-1β (IL-1β, which is secreted mainly from macrophages upon cytosolic stimuli by activating cysteine protease caspase-1 within a complex called the inflammasome, and is a key player in initiating and maintaining the inflammatory response. However, the mechanism for IL-1β maturation upon O. tsutsugamushi infection has not been identified. In this study, we show that IL-1 receptor signaling is required for efficient host protection from O. tsutsugamushi infection. Live Orientia, but not heat- or UV-inactivated Orientia, activates the inflammasome through active bacterial uptake and endo/phagosomal maturation. Furthermore, Orientia-stimulated secretion of IL-1β and activation of caspase-1 are ASC- and caspase-1- dependent since IL-1β production was impaired in Asc- and caspase-1-deficient macrophages but not in Nlrp3-, Nlrc4- and Aim2-deficient macrophages. Therefore, live O. tsutsugamushi triggers ASC inflammasome activation leading to IL-1β production, which is a critical innate immune response for effective host defense.

Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides.

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Lee, Soo Chan; Li, Alicia; Calo, Silvia; Inoue, Makoto; Tonthat, Nam K; Bain, Judith M; Louw, Johanna; Shinohara, Mari L; Erwig, Lars P; Schumacher, Maria A; Ko, Dennis C; Heitman, Joseph

2015-09-01

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi. © 2015 John Wiley & Sons Ltd.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

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Prajna Jena

Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

S1PR3 Signaling Drives Bacterial Killing and Is Required for Survival in Bacterial Sepsis.

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Hou, JinChao; Chen, QiXing; Wu, XiaoLiang; Zhao, DongYan; Reuveni, Hadas; Licht, Tamar; Xu, MengLong; Hu, Hu; Hoeft, Andreas; Ben-Sasson, Shmuel A; Shu, Qiang; Fang, XiangMing

2017-12-15

Efficient elimination of pathogenic bacteria is a critical determinant in the outcome of sepsis. Sphingosine-1-phosphate receptor 3 (S1PR3) mediates multiple aspects of the inflammatory response during sepsis, but whether S1PR3 signaling is necessary for eliminating the invading pathogens remains unknown. To investigate the role of S1PR3 in antibacterial immunity during sepsis. Loss- and gain-of-function experiments were performed using cell and murine models. S1PR3 levels were determined in patients with sepsis and healthy volunteers. S1PR3 protein levels were up-regulated in macrophages upon bacterial stimulation. S1pr3 -/- mice showed increased mortality and increased bacterial burden in multiple models of sepsis. The transfer of wild-type bone marrow-derived macrophages rescued S1pr3 -/- mice from lethal sepsis. S1PR3-overexpressing macrophages further ameliorated the mortality rate of sepsis. Loss of S1PR3 led to markedly decreased bacterial killing in macrophages. Enhancing endogenous S1PR3 activity using a peptide agonist potentiated the macrophage bactericidal function and improved survival rates in multiple models of sepsis. Mechanically, the reactive oxygen species levels were decreased and phagosome maturation was delayed in S1pr3 -/- macrophages due to impaired recruitment of vacuolar protein-sorting 34 to the phagosomes. In addition, S1RP3 expression levels were elevated in monocytes from patients with sepsis. Higher levels of monocytic S1PR3 were associated with efficient intracellular bactericidal activity, better immune status, and preferable outcomes. S1PR3 signaling drives bacterial killing and is essential for survival in bacterial sepsis. Interventions targeting S1PR3 signaling could have translational implications for manipulating the innate immune response to combat pathogens.

Leghemoglobin-derived radicals. Evidence for multiple protein-derived radicals and the initiation of peribacteroid membrane damage

DEFF Research Database (Denmark)

Moreau, S; Davies, Michael Jonathan; Mathieu, C

1996-01-01

, with the consequent generation of lipid-derived radicals. The formation of such radicals may result in the depletion of membrane antioxidants and the initiation of lipid peroxidation. This transfer of damage from the heme center via the protein surface to neighboring membranes may be of considerable biological......-derived phenoxyl radical present at Tyr-133 in the soybean protein and Tyr-138 in the lupin protein. To obtain further information on these protein radicals and their potential interaction with the physiologically important peribacteroid membrane (which surrounds the microsymbiont in vivo), EPR spin trapping......); these radicals may be side chain- or alpha-carbon-derived, their exact sites have not been determined. Some of these radicals are on the protein surface and may be key intermediates in the formation of protein dimers. These radicals have been shown to be capable of reacting with peribacteroid membrane fractions...

Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro

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Sophia C. Dudte

2017-08-01

Full Text Available Yersinia pestis is a gram-negative, zoonotic, bacterial pathogen, and the causative agent of plague. The bubonic form of plague occurs subsequent to deposition of bacteria in the skin by the bite of an infected flea. Neutrophils are recruited to the site of infection within the first few hours and interactions between neutrophils and Y. pestis have been demonstrated in vivo. In contrast to macrophages, neutrophils have been considered non-permissive to Y. pestis intracellular survival. Several studies have shown killing of the vast majority of Y. pestis ingested by human neutrophils. However, survival of 10–15% of Y. pestis after phagocytosis by neutrophils is consistently observed. Furthermore, these surviving bacteria eventually replicate within and escape from the neutrophils. We set out to further characterize the interactions between Y. pestis and human neutrophils by (1 determining the effects of known Y. pestis virulence factors on bacterial survival after uptake by neutrophils, (2 examining the mechanisms employed by the neutrophil to kill the majority of intracellular Y. pestis, (3 determining the activation phenotype of Y. pestis-infected neutrophils, and (4 characterizing the Y. pestis-containing phagosome in neutrophils. We infected human neutrophils in vitro with Y. pestis and assayed bacterial survival and uptake. Deletion of the caf1 gene responsible for F1 capsule production resulted in significantly increased uptake of Y. pestis. Surprisingly, while the two-component regulator PhoPQ system is important for survival of Y. pestis within neutrophils, pre-induction of this system prior to infection did not increase bacterial survival. We used an IPTG-inducible mCherry construct to distinguish viable from non-viable intracellular bacteria and determined the association of the Y. pestis-containing phagosome with neutrophil NADPH-oxidase and markers of primary, secondary and tertiary granules. Additionally, we show that inhibition of

Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

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Tame, Akihiro; Yoshida, Takao; Ohishi, Kazue; Maruyama, Tadashi

2015-07-01

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 μm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 μm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 μm) and contained small basophilic granules (diameter, 0.3-0.4 μm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 μm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

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van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

Precursors-Derived Ceramic Membranes for High-Temperature Separation of Hydrogen

OpenAIRE

Yuji, Iwamoto

2007-01-01

This review describes recent progress in the development of hydrogen-permselective ceramic membranes derived from organometallic precursors. Microstructure and gas transport property of microporous amorphous silica-based membranes are briefly described. Then, high-temperature hydrogen permselectivity, hydrothermal stability as well as hydrogen/steam selectivity of the amorphous silica-based membranes are discussed from a viewpoint of application to membrane reactors for conversion enhancement...

Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages.

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Tanigawa, Kazunari; Degang, Yang; Kawashima, Akira; Akama, Takeshi; Yoshihara, Aya; Ishido, Yuko; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

2012-05-01

Mycobacterium leprae (M. leprae), the causative agent of leprosy, parasitizes within the foamy or enlarged phagosome of macrophages where rich lipids accumulate. Although the mechanisms for lipid accumulation in the phagosome have been clarified, it is still unclear how such large amounts of lipids escape degradation. To further explore underlying mechanisms involved in lipid catabolism in M. leprae-infected host cells, we examined the expression of hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization and lipolysis, in human macrophage THP-1 cells. We found that infection by live M. leprae significantly suppressed HSL expression levels. This suppression was not observed with dead M. leprae or latex beads. Macrophage activation by peptidoglycan (PGN), the ligand for toll-like receptor 2 (TLR2), increased HSL expression; however, live M. leprae suppressed this increase. HSL expression was abolished in the slit-skin smear specimens from patients with lepromatous and borderline leprosy. In addition, the recovery of HSL expression was observed in patients who experienced a lepra reaction, which is a cell-mediated, delayed-type hypersensitivity immune response, or in patients who were successfully treated with multi-drug therapy. These results suggest that M. leprae suppresses lipid degradation through inhibition of HSL expression, and that the monitoring of HSL mRNA levels in slit-skin smear specimens may be a useful indicator of patient prognosis.

Inhaled Cadmium Oxide Nanoparticles: Their in Vivo Fate and Effect on Target Organs

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Jana Dumkova

2016-06-01

Full Text Available The increasing amount of heavy metals used in manufacturing equivalently increases hazards of environmental pollution by industrial products such as cadmium oxide (CdO nanoparticles. Here, we aimed to unravel the CdO nanoparticle destiny upon their entry into lungs by inhalations, with the main focus on the ultrastructural changes that the nanoparticles may cause to tissues of the primary and secondary target organs. We indeed found the CdO nanoparticles to be transported from the lungs into secondary target organs by blood. In lungs, inhaled CdO nanoparticles caused significant alterations in parenchyma tissue including hyperemia, enlarged pulmonary septa, congested capillaries, alveolar emphysema and small areas of atelectasis. Nanoparticles were observed in the cytoplasm of cells lining bronchioles, in the alveolar spaces as well as inside the membranous pneumocytes and in phagosomes of lung macrophages. Nanoparticles even penetrated through the membrane into some organelles including mitochondria and they also accumulated in the cytoplasmic vesicles. In livers, inhalation caused periportal inflammation and local hepatic necrosis. Only minor changes such as diffusely thickened filtration membrane with intramembranous electron dense deposits were observed in kidney. Taken together, inhaled CdO nanoparticles not only accumulated in lungs but they were also transported to other organs causing serious damage at tissue as well as cellular level.

Inhaled Cadmium Oxide Nanoparticles: Their in Vivo Fate and Effect on Target Organs.

Science.gov (United States)

Dumkova, Jana; Vrlikova, Lucie; Vecera, Zbynek; Putnova, Barbora; Docekal, Bohumil; Mikuska, Pavel; Fictum, Petr; Hampl, Ales; Buchtova, Marcela

2016-06-03

The increasing amount of heavy metals used in manufacturing equivalently increases hazards of environmental pollution by industrial products such as cadmium oxide (CdO) nanoparticles. Here, we aimed to unravel the CdO nanoparticle destiny upon their entry into lungs by inhalations, with the main focus on the ultrastructural changes that the nanoparticles may cause to tissues of the primary and secondary target organs. We indeed found the CdO nanoparticles to be transported from the lungs into secondary target organs by blood. In lungs, inhaled CdO nanoparticles caused significant alterations in parenchyma tissue including hyperemia, enlarged pulmonary septa, congested capillaries, alveolar emphysema and small areas of atelectasis. Nanoparticles were observed in the cytoplasm of cells lining bronchioles, in the alveolar spaces as well as inside the membranous pneumocytes and in phagosomes of lung macrophages. Nanoparticles even penetrated through the membrane into some organelles including mitochondria and they also accumulated in the cytoplasmic vesicles. In livers, inhalation caused periportal inflammation and local hepatic necrosis. Only minor changes such as diffusely thickened filtration membrane with intramembranous electron dense deposits were observed in kidney. Taken together, inhaled CdO nanoparticles not only accumulated in lungs but they were also transported to other organs causing serious damage at tissue as well as cellular level.

Efecto de la fagocitosis, la infección po Leishmania amazonenesis y Leshmania braziliensis sobre la distribción de filamentos de actina y microtúbulos en macrófagos murinos

OpenAIRE

Chacón Rojas, Marcos Nicolás

2011-01-01

Por inmunofluorescencia se estudió la distribución del citoesqueleto en macrófagos activados, con fagosomas de diferentes tamaños e infectados con Leishmania. También se evaluaron los niveles de actina y tubulina en los mismos grupos. Se encontró que fagosomas grandes alteran la distribución del citoesqueleto sin afectar niveles de actina y tubulina. / Abstract. Cytoskeleton distribution was studied in macrophages activated, with phagosomes of different size and infected with Leishmania pa...

Ultrastructure of photo-sensory cells and pigment epithelium in the retina of the Antarctic fish Notothenia neglecta Nybelin (Nototheniidae

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Lucelia Donatti

2002-03-01

Full Text Available The Antarctic nototheniid Notothenia neglecta is the dominant fish in its habitat in Admiralty Bay, King George Island. They are predators, often ambush feeders, with accurate visual behaviour. For that reason, the ultrastructure of retinal photoreceptive cells and the pigment epithelium was analysed through electron microscopy. Their retina has a pigment epithelium, five different photoreceptors : rods, short single, long single, double, and triple cones, and neurones and support cells. The pigment epithelium is characterised by infoldings of the basal membrane, basal mitochondria, smooth reticule, large amount of microtubules, melanin granules, phagosomes and detached membranes of photoreceptors. Cones show bimembranous discs in the outer segment, an accessory outer segment, a connecting cilium, calycal processes, microtubules in the inferior ellipsoid and myoid, centrioles in the ellipsoid, interdigitating myoid fins and apical microvilli of Muller cells in the myoid and elliposid region. All these features allow all sorts of adaptations to the environmental photic variations, and situate N. neglecta among fish with a complex retina, with cells that are arranged in ten layers, allowing horizontal and vertical integration among them. This allows optimal visual behaviour and perception of food and environment in every Antarctic season.

Newly synthesized benzanthrone derivatives as prospective fluorescent membrane probes

International Nuclear Information System (INIS)

Zhytniakivska, Olga; Trusova, Valeriya; Gorbenko, Galyna; Kirilova, Elena; Kalnina, Inta; Kirilov, Georgiy; Kinnunen, Paavo

2014-01-01

Fluorescence spectral properties of a series of novel benzanthrone derivatives have been explored in lipid bilayers composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) and anionic phospholipid cardiolipin (CL). Analysis of partition coefficients showed that all the examined compounds possess rather high lipid-associating ability, with the amidino derivatives exhibiting stronger membrane partitioning compared with the aminobenzanthrones. To understand how benzanthrone partition properties correlate with their structure, quantitative structure property relationship (QSPR) analysis was performed involving a range of quantum chemical molecular descriptors. -- Highlights: • Benzanthrone partitioning into lipid bilayer correlates with lipophilicity of the dyes. • Partition properties of benzanthrones depend on the dye dipole moment. • Amidino derivatives exhibit higher membrane affinity than aminobenzanthrones

Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: A non-conventional hemolysin and a ribosomal RNA methyl transferase

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Ahmed Neesar

2010-09-01

Full Text Available Abstract Background Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein. Results The recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s. The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth

DMPD: Linking inflammasome activation and phagosome maturation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available ml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary

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Sandy B. Serizier

2017-11-01

Full Text Available For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells. Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/β-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary.

Science.gov (United States)

Serizier, Sandy B; McCall, Kimberly

2017-01-01

For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells). Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/β-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

Science.gov (United States)

Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

2013-01-01

Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

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Jinli Wang

Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

The LRRC8A Mediated “Swell Activated” Chloride Conductance Is Dispensable for Vacuolar Homeostasis in Neutrophils

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Philippe Behe

2017-05-01

Full Text Available The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl- current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A; the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their “swell activated” Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

Extraction Socket Preservation Using Porcine-Derived Collagen Membrane Alone or Associated with Porcine-Derived Bone. Clinical Results of Randomized Controlled Study

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Renzo Guarnieri

2017-03-01

Full Text Available Objectives: The aim of present randomized controlled clinical trial was to clinically evaluate hard tissue changes after extraction socket preservation procedures compared to natural spontaneous healing. Material and Methods: Thirty patients were enrolled in the present study and underwent single-tooth extraction in the premolar/molar areas. Ten sites were grafted with porcine-derived bone covered by collagen membrane, 10 covered by porcine-derived collagen membrane alone, and 10 underwent natural spontaneous healing. Vertical and horizontal bone changes after 3-month were evaluated at implant placement. Results: The vertical and horizontal bone changes at the extraction sockets treated with collagen membrane alone (vertical: -0.55 [SD 0.11] mm, and horizontal: -1.21 [SD 0.69] mm and collagen membrane plus porcine-derived bone (vertical: -0.37 [SD 0.7] mm, and horizontal: -0.91 [SD 0.53] mm were found significantly lower (P < 0.001, when compared to non-grafted sockets (vertical: -2.09 [SD 0.19] mm, and horizontal: -3.96 [SD 0.87] mm. In type 1 extraction sockets, in premolar sites, and in presence of vestibular bone thicknesses ≥ 1.5 mm, the use of collagen membrane alone revealed similar outcomes to those with additional graft material. Conclusions: At the re-entry surgery, extraction sockets grafted with porcine-derived bone and covered by collagen membrane, and extraction sockets covered by porcine-derived collagen membrane alone, showed significantly lower vertical and horizontal bone changes, compared to extraction sockets sites underwent natural spontaneous healing. However, a complete prevention of remodelling is not achievable, irrespective of the technique used.

A review of recent advances in molecular simulation of graphene-derived membranes for gas separation

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Fatemi, Seyyed Mahmood; Abbasi, Zeynab; Rajabzadeh, Halimeh; Hashemizadeh, Seyyed Ali; Deldar, Amir Noori

2017-07-01

To obtain an ideal membrane for gas separation the following three characteristics should be considered: the membrane should be as thin as possible, be mechanically robust, and have well-defined pore sizes. These features will maximize its solvent flux, preserve it from fracture, and guarantee its selectivity. These attractive properties of graphene-derived membranes introduce them as appropriate candidates for gas separation and gas molecular-sieving processes in nanoscale dimensions. The current effort has focused on two issues, including the review of the most newly progression on drilling holes in single graphene membranes for making ultrathin membranes for gas separation, and studying functionalized nanoporous sheet and graphene-derived membranes, including doped graphene, graphene oxide, fluorographene, and reduced graphene oxide from theoretical perspectives for making functional coatings for nano ultrafiltration for gas separation. We investigated the basic mechanism of separation by membranes derived from graphene and relevant possible applications. Functionalized nanoporous membranes as novel approach are characterized by low energy cost in realizing high throughput molecular-sieving separation.

Enhanced biofouling resistance of polyethersulfone membrane surface modified with capsaicin derivative and itaconic acid

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Wang, Jian [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China); Gao, Xueli, E-mail: gxl_ouc@126.com [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China); Wang, Qun; Sun, Haijing; Wang, Xiaojuan [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China); Gao, Congjie, E-mail: gaocjie@ouc.edu.cn [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China)

2015-11-30

Graphical abstract: - Highlights: • PES membrane was modified with a capsaicin derivative. • UV-assisted graft polymerization was carried out on membrane surface. • The capsaicin derivative modified membrane shows better antibiofouling property. - Abstract: The culprit of biofouling is the reproduction of viable microorganisms on the membrane surface. Recently, functionalization of membrane surface with natural antibacterial agents has drawn great attention. This work presents the fabrication of antibiofouling polyethersulfone (PES) ultrafiltration (UF) membranes by UV-assisted photo grafting of capsaicin derivative (N-(4-hydroxy-3-methoxy-benzyl)-acrylamide, HMBA) and itaconic acid (IA) on the surface of PES membrane. Results of FTIR-ATR, water static contact angle (WSCA) and atomic force microscopy (AFM) analysis confirmed the successful grafting of HMBA and IA on the membrane surface. We investigated the antifouling and antibacterial properties of these membranes using BSA and Escherichia coli as the test model, respectively. During a 150-min test, the modified membranes show much lower flux decline (42.7% for PES-g-1H0I, 22.2% for PES-g-1H1I and 7.7% for PES-g-1H5I) when compared with the pristine membrane (flux declined by 77%). The modified membranes exhibit excellent antibacterial activity (nearly 100%) when UV irradiation time was 6 min. The morphological study suggested that the E. coli on the pristine membrane showed a regular and smooth surface while that on the modified membrane was disrupted, which validated the antibacterial activity of the modified membranes.

Enhanced biofouling resistance of polyethersulfone membrane surface modified with capsaicin derivative and itaconic acid

International Nuclear Information System (INIS)

Wang, Jian; Gao, Xueli; Wang, Qun; Sun, Haijing; Wang, Xiaojuan; Gao, Congjie

2015-01-01

Graphical abstract: - Highlights: • PES membrane was modified with a capsaicin derivative. • UV-assisted graft polymerization was carried out on membrane surface. • The capsaicin derivative modified membrane shows better antibiofouling property. - Abstract: The culprit of biofouling is the reproduction of viable microorganisms on the membrane surface. Recently, functionalization of membrane surface with natural antibacterial agents has drawn great attention. This work presents the fabrication of antibiofouling polyethersulfone (PES) ultrafiltration (UF) membranes by UV-assisted photo grafting of capsaicin derivative (N-(4-hydroxy-3-methoxy-benzyl)-acrylamide, HMBA) and itaconic acid (IA) on the surface of PES membrane. Results of FTIR-ATR, water static contact angle (WSCA) and atomic force microscopy (AFM) analysis confirmed the successful grafting of HMBA and IA on the membrane surface. We investigated the antifouling and antibacterial properties of these membranes using BSA and Escherichia coli as the test model, respectively. During a 150-min test, the modified membranes show much lower flux decline (42.7% for PES-g-1H0I, 22.2% for PES-g-1H1I and 7.7% for PES-g-1H5I) when compared with the pristine membrane (flux declined by 77%). The modified membranes exhibit excellent antibacterial activity (nearly 100%) when UV irradiation time was 6 min. The morphological study suggested that the E. coli on the pristine membrane showed a regular and smooth surface while that on the modified membrane was disrupted, which validated the antibacterial activity of the modified membranes.

Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

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Martins, Danubia Batista; Nasário, Fábio Domingues; Silva-Gonçalves, Laiz Costa; de Oliveira Tiera, Vera Aparecida; Arcisio-Miranda, Manoel; Tiera, Marcio José; Dos Santos Cabrera, Marcia Perez

2018-02-01

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism.

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Solomonson, Matthew; Setiaputra, Dheva; Makepeace, Karl A T; Lameignere, Emilie; Petrotchenko, Evgeniy V; Conrady, Deborah G; Bergeron, Julien R; Vuckovic, Marija; DiMaio, Frank; Borchers, Christoph H; Yip, Calvin K; Strynadka, Natalie C J

2015-03-03

Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interaction of MreB-derived antimicrobial peptides with membranes.

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Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

From the Outside-In: the Francisella tularensis Envelope and Virulence

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Hannah M. Rowe

2015-12-01

Full Text Available Francisella tularensis is a highly-infectious bacterium that causes the rapid, and often lethal disease, tularemia. Many studies have been performed to identify and characterize the virulence factors that F. tularensis uses to infect a wide variety of hosts and host cell types, evade immune defenses, and induce severe disease and death. This review focuses on the virulence factors that are present in the F. tularensis envelope, including capsule, LPS, outer membrane, periplasm, inner membrane, secretion systems, and various molecules in each of aforementioned sub-compartments. Whereas no single bacterial molecule or molecular complex single-handedly controls F. tularensis virulence, we review here how diverse bacterial systems work in conjunction to subvert the immune system, attach to and invade host cells, alter phagosome/lysosome maturation pathways, replicate in host cells without being detected, inhibit apoptosis, and induce host cell death for bacterial release and infection of adjacent cells. Given that the F. tularensis envelope is the outermost layer of the bacterium, we highlight herein how many of these molecules directly interact with the host to promote infection and disease. These and future envelope studies are important to advance our collective understanding of F. tularensis virulence mechanisms and offer targets for future vaccine development efforts.

Platinum-Based Drugs Differentially Affect the Ultrastructure of Breast Cancer Cell Types

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Shadia Al-Bahlani

2017-01-01

Full Text Available Breast cancer (BC is the most common cause of cancer-related death worldwide. Although platinum-based drugs (PBDs are effective anticancer agents, responsive patients eventually become resistant. While resistance of some cancers to PBDs has been explored, the cellular responses of BC cells are not studied yet. Therefore, we aim to assess the differential effects of PBDs on BC ultrastructure. Three representative cells were treated with different concentrations and timing of Cisplatin, Carboplatin, and Oxaliplatin. Changes on cell surface and ultrastructure were detected by scanning (SEM and transmission electron microscope (TEM. In SEM, control cells were semiflattened containing microvilli with extending lamellipodia while treated ones were round with irregular surface and several pores, indicating drug entry. Prolonged treatment resembled distinct apoptotic features such as shrinkage, membrane blebs, and narrowing of lamellipodia with blunt microvilli. TEM detected PBDs’ deposits that scattered among cellular organelles inducing structural distortion, lumen swelling, chromatin condensation, and nuclear fragmentation. Deposits were attracted to fat droplets, explained by drug hydrophobic properties, while later they were located close to cell membrane, suggesting drug efflux. Phagosomes with destructed organelles and deposits were detected as defending mechanism. Understanding BC cells response to PBDs might provide new insight for an effective treatment.

Autophagy in C. elegans development.

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Palmisano, Nicholas J; Meléndez, Alicia

2018-04-27

Autophagy involves the sequestration of cytoplasmic contents in a double-membrane structure referred to as the autophagosome and the degradation of its contents upon delivery to lysosomes. Autophagy activity has a role in multiple biological processes during the development of the nematode Caenorhabditis elegans. Basal levels of autophagy are required to remove aggregate prone proteins, paternal mitochondria, and spermatid-specific membranous organelles. During larval development, autophagy is required for the remodeling that occurs during dauer development, and autophagy can selectively degrade components of the miRNA-induced silencing complex, and modulate miRNA-mediated silencing. Basal levels of autophagy are important in synapse formation and in the germ line, to promote the proliferation of proliferating stem cells. Autophagy activity is also required for the efficient removal of apoptotic cell corpses by promoting phagosome maturation. Finally, autophagy is also involved in lipid homeostasis and in the aging process. In this review, we first describe the molecular complexes involved in the process of autophagy, its regulation, and mechanisms for cargo recognition. In the second section, we discuss the developmental contexts where autophagy has been shown to be important. Studies in C. elegans provide valuable insights into the physiological relevance of this process during metazoan development. Copyright © 2018 Elsevier Inc. All rights reserved.

Subcellular localization of ammonium transporters in Dictyostelium discoideum

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Davis Carter T

2008-12-01

Full Text Available Abstract Background With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. Results Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. Conclusion Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

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Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

KAUST Repository

Zhu, Jiang; Zhang, Huoming; Guo, Tiannan; Li, Wenying; Li, Huiyu; Zhu, Yi; Huang, Shiang

2014-01-01

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

KAUST Repository

Zhu, Jiang

2014-06-11

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

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Abirami Kugadas

2016-08-01

Full Text Available AbstractThe ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP, the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophage and MAC-T cells and coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increase bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5 conditions. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

In Vivo Immunotoxicity of SiO2@(Y0.5Gd0.45Eu0.052O3 as Dual-Modality Nanoprobes

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Xiumei Tian

2014-08-01

Full Text Available We have successfully synthesized SiO2@(Y0.5Gd0.45Eu0.052O3 nanocomposites as a potential dual-modality nanoprobe for molecular imaging in vitro. However, their immunotoxicity assessment in vivo remains unknown. In this article, the in vitro biocompatibility of our dual-modality nanoprobes was assayed in terms of cell viability and apoptosis. In vivo immunotoxicity was investigated by monitoring the generation of reactive oxygen species (ROS, cluster of differentiation (CD markers and cytokines in Balb/c mice. The data show that the in vitro biocompatibility was satisfactory. In addition, the immunotoxicity data revealed there are no significant changes in the expression levels of CD11b and CD71 between the nanoprobe group and the Gd in a diethylenetriaminepentaacetic acid (DTPA chelator (Gd-DTPA group 24 h after injection in Balb/c mice (p > 0.05. Importantly, there are significant differences in the expression levels of CD206 and CD25 as well as the secretion of IL-4 and the generation of ROS 24 h after injection (p < 0.05. Transmission electron microscopy (TEM images showed that few nanoprobes were localized in the phagosomes of liver and lung. In conclusion, the toxic effects of our nanoprobes may mainly result from the aggregation of particles in phagosomes. This accumulation may damage the microstructure of the cells and generate oxidative stress reactions that further stimulate the immune response. Therefore, it is important to evaluate the in vivo immunotoxicity of these rare earth-based biomaterials at the molecular level before molecular imaging in vivo.

Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy

NARCIS (Netherlands)

Surewaard, Bas G.J.; Kubes, Paul

2017-01-01

It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium.

Science.gov (United States)

Silva, Tânia; Moreira, Ana C; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

2017-01-01

Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17-30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17-30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17-30 did not localize to M. avium -harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17-30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis , M. leprae , M. avium , etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we

Translocation mechanism of C{sub 60} and C{sub 60} derivations across a cell membrane

Energy Technology Data Exchange (ETDEWEB)

Liang, Lijun, E-mail: michael.lijunl@gmail.com [Hangzhou Dianzi University, College of Life Information Science and Instrument Engineering (China); Kang, Zhengzhong [Zhejiang University, Department of Chemistry (China); Shen, Jia-Wei, E-mail: shen.jiawei@hotmail.com [Hangzhou Normal University, School of Medicine (China)

2016-11-15

Carbon-based nanoparticles (NPs) such as fullerenes and nanotubes have been extensively studied for drug delivery in recent years. The permeation process of fullerene and its derivative molecules through membrane is essential to the utilization of fullerene-based drug delivery system, but the mechanism and the dynamics of permeation through cell membrane are still unclear. In this study, coarse-grained molecular dynamics simulations were performed to investigate the permeation process of functionalized fullerene molecules (ca. 0.72 nm) through the membrane. Our results show that single functionalized fullerene molecule in such nanoscale could permeate the lipid membrane in micro-second time scale. Pristine C{sub 60} molecules prefer to aggregate into several small clusters while C{sub 60}OH{sub 15} molecules could aggregate into one big cluster to permeate through the lipid membrane. After permeation of C{sub 60} or its derivatives into membrane, all C{sub 60} and C{sub 60}OH{sub 15} molecules disaggregated and monodispersed in the lipid membrane.

Host and bacterial proteins that repress recruitment of LC3 to Shigella early during infection.

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Leigh A Baxt

Full Text Available Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min., the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4-6 hr by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG. Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

Directory of Open Access Journals (Sweden)

Mamata Gurung

Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

Novel polybenzimidazole derivatives for high temperature polymer electrolyte membrane fuel cell applications

Science.gov (United States)

Xiao, Lixiang

Recent advances have made polymer electrolyte membrane fuel cells (PEMFCs) a leading alternative to internal combustion engines for both stationary and transportation applications. In particular, high temperature polymer electrolyte membranes operational above 120°C without humidification offer many advantages including fast electrode kinetics, high tolerance to fuel impurities and simple thermal and water management systems. A series of polybenzimidazole (PBI) derivatives including pyridine-based PBI (PPBI) and sulfonated PBI (SPBI) homopolymers and copolymers have been synthesized using polyphosphoric acid (PPA) as both solvent and polycondensation agent. High molecular weight PBI derivative polymers were obtained with well controlled backbone structures in terms of pyridine ring content, polymer backbone rigidity and degree of sulfonation. A novel process, termed the PPA process, has been developed to prepare phosphoric acid (PA) doped PBI membranes by direct-casting of the PPA polymerization solution without isolation or re-dissolution of the polymers. The subsequent hydrolysis of PPA to PA by moisture absorbed from the atmosphere usually induced a transition from the solution-like state to a gel-like state and produced PA doped PBI membranes with a desirable suite of physiochemical properties characterized by the PA doping levels, mechanical properties and proton conductivities. The effects of the polymer backbone structure on the polymer characteristics and membrane properties, i.e., the structure-property relationships of the PBI derivative polymers have been studied. The incorporation of additional basic nitrogen containing pyridine rings and sulfonic acid groups enhanced the polymer solubility in acid and dipolar solvents while retaining the inherently high thermal stability of the PBI heteroaromatic backbone. In particular, the degradation of the SPBI polymers with reasonable high molecular weights commenced above 450°C, notably higher than other

Pathogenicity of Shigella in chickens.

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Shi, Run; Yang, Xia; Chen, Lu; Chang, Hong-tao; Liu, Hong-ying; Zhao, Jun; Wang, Xin-wei; Wang, Chuan-qing

2014-01-01

Shigellosis in chickens was first reported in 2004. This study aimed to determine the pathogenicity of Shigella in chickens and the possibility of cross-infection between humans and chickens. The pathogenicity of Shigella in chickens was examined via infection of three-day-old SPF chickens with Shigella strain ZD02 isolated from a human patient. The virulence and invasiveness were examined by infection of the chicken intestines and primary chicken intestinal epithelial cells. The results showed Shigella can cause death via intraperitoneal injection in SPF chickens, but only induce depression via crop injection. Immunohistochemistry and transmission electron microscopy revealed the Shigella can invade the intestinal epithelia. Immunohistochemistry of the primary chicken intestinal epithelial cells infected with Shigella showed the bacteria were internalized into the epithelial cells. Electron microscopy also confirmed that Shigella invaded primary chicken intestinal epithelia and was encapsulated by phagosome-like membranes. Our data demonstrate that Shigella can invade primary chicken intestinal epithelial cells in vitro and chicken intestinal mucosa in vivo, resulting in pathogenicity and even death. The findings suggest Shigella isolated from human or chicken share similar pathogenicity as well as the possibility of human-poultry cross-infection, which is of public health significance.

Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol-soluble modulin α

NARCIS (Netherlands)

Grosz, Magdalena; Kolter, Julia; Paprotka, Kerstin; Winkler, Ann-Cathrin; Schäfer, Daniel; Chatterjee, Som Subra; Geiger, Tobias; Wolz, Christiane; Ohlsen, Knut; Otto, Michael; Rudel, Thomas; Sinha, Bhanu; Fraunholz, Martin

Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of

Physiochemical properties and resorption progress of porcine skin-derived collagen membranes: In vitro and in vivo analysis.

Science.gov (United States)

An, Yin-Zhe; Kim, You-Kyoung; Lim, Su-Min; Heo, Yeong-Ku; Kwon, Mi-Kyung; Cha, Jae-Kook; Lee, Jung-Seok; Jung, Ui-Won; Choi, Seong-Ho

2018-03-30

The aim of the present study was to evaluate the physiochemical properties and resorption progress of two cross-linked, porcine skin-derived collagen membranes and compare their features with those of a membrane without cross-linking (Bio-Gide ® [BG], Geistlich Biomaterials, Wolhusen, Switzerland). Three porcine skin-derived collagen membranes, dehydrothermally (DHT) cross-linked (experimental), DHT and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (DHT/EDC) cross-linked (experimental) and BG were investigated for their morphology, enzyme resistance, and tensile strength in vitro and biodegradation in vivo. DHT and DHT/EDC membranes exhibited irregular, interconnected macro- and micropores that formed a 3D mesh, whereas BG exhibited individual collagen fibrils interlaced to form coarse collagen strands. In enzyme resistance and tensile strength tests, DHT and DHT/EDC membranes demonstrated good resistance and mechanical properties compared with BG. In vivo, all three membranes were well integrated into the surrounding connective tissue. Thus, the DHT membrane exhibited its potential as a barrier membrane for guided bone and tissue regeneration.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

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Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

2012-08-01

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

Long-term smoking alters abundance of over half of the proteome in bronchoalveolar lavage cell in smokers with normal spirometry, with effects on molecular pathways associated with COPD.

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Yang, Mingxing; Kohler, Maxie; Heyder, Tina; Forsslund, Helena; Garberg, Hilde K; Karimi, Reza; Grunewald, Johan; Berven, Frode S; Magnus Sköld, C; Wheelock, Åsa M

2018-03-08

Smoking represents a significant risk factor for many chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). To identify dysregulation of specific proteins and pathways in bronchoalveolar lavage (BAL) cells associated with smoking, isobaric tags for relative and absolute quantitation (iTRAQ)-based shotgun proteomics analyses were performed on BAL cells from healthy never-smokers and smokers with normal lung function from the Karolinska COSMIC cohort. Multivariate statistical modeling, multivariate correlations with clinical data, and pathway enrichment analysis were performed. Smoking exerted a significant impact on the BAL cell proteome, with more than 500 proteins representing 15 molecular pathways altered due to smoking. The majority of these alterations occurred in a gender-independent manner. The phagosomal- and leukocyte trans endothelial migration (LTM) pathways significantly correlated with FEV 1 /FVC as well as the percentage of CD8 + T-cells and CD8 + CD69 + T-cells in smokers. The correlations to clinical parameters in healthy never-smokers were minor. The significant correlations of proteins in the phagosome- and LTM pathways with activated cytotoxic T-cells (CD69+) and the level of airway obstruction (FEV 1 /FVC) in smokers, both hallmarks of COPD, suggests that these two pathways may play a role in the molecular events preceding the development of COPD in susceptible smokers. Both pathways were found to be further dysregulated in COPD patients from the same cohort, thereby providing further support to this hypothesis. Given that not all smokers develop COPD in spite of decades of smoking, it is also plausible that some of the molecular pathways associated with response to smoking exert protective mechanisms to smoking-related pathologies in resilient individuals. ClinicalTrials.gov identifier NCT02627872 ; Retrospectively registered on December 9, 2015.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

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Fineran, Paul; Lloyd-Evans, Emyr; Lack, Nathan A; Platt, Nick; Davis, Lianne C; Morgan, Anthony J; Höglinger, Doris; Tatituri, Raju Venkata V; Clark, Simon; Williams, Ian M; Tynan, Patricia; Al Eisa, Nada; Nazarova, Evgeniya; Williams, Ann; Galione, Antony; Ory, Daniel S; Besra, Gurdyal S; Russell, David G; Brenner, Michael B; Sim, Edith; Platt, Frances M

2016-11-18

Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis , achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria

A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

Energy Technology Data Exchange (ETDEWEB)

Mondal, Tapan Kumar; Emeny, Rebecca T.; Gao, Donghong; Ault, Jeffrey G.; Kasten-Jolly, Jane; Lawrence, David A., E-mail: david.lawrence@health.ny.gov

2015-12-01

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2 h, host defenses against bacterial growth are suppressed 2–3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidation (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes. - Highlights: • Cold-restraint (physical/psychological stress) induces early oxidative stress. • The oxidative stress relates to catecholamine signaling beta-adrenoceptors. • Physical/psychological stress combines infection enhancing inflammation. • Endoplasmic reticulum

Staphylococcus aureus ?-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

OpenAIRE

Thay, Bernard; Wai, Sun Nyunt; Oscarsson, Jan

2013-01-01

Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol...

Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

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Wavre-Shapton, Silène T; Tolmachova, Tanya; Lopes da Silva, Mafalda; da Silva, Mafalda Lopes; Futter, Clare E; Seabra, Miguel C

2013-01-01

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

Directory of Open Access Journals (Sweden)

Silène T Wavre-Shapton

Full Text Available The retinal pigment epithelium (RPE is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1. REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox, Tyr-Cre+. Transmission electron microscopy (TEM was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

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Anahí Capmany

2010-11-01

Full Text Available Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

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Capmany, Anahí; Damiani, María Teresa

2010-11-22

Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

Bone Morphometric Evaluation around Immediately Placed Implants Covered with Porcine-Derived Pericardium Membrane: An Experimental Study in Dogs

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Ryo Jimbo

2012-01-01

Full Text Available Objective. To investigate whether porcine-derived bioresorbable pericardium membrane coverage enhances the osseointegration around implants placed in fresh extraction sockets. Study Design. Twenty-four commercially available endosseous implants were placed in the fresh extraction sockets of the mandibular first molar of mature beagles (. On one side, implants and osteotomy sites were covered with porcine-derived bioresorbable pericardium membranes, whereas on the other side, no membranes were used. After 6 weeks, samples were retrieved and were histologically processed for histomorphometric analysis. Results. The histological observation showed that bone loss and soft tissue migration in the coronal region of the implant were evident for the control group, whereas bone fill was evident up to the neck of the implant for the membrane-covered group. Bone-to-implant contact was significantly higher for the membrane-covered group compared to the control group, 75% and 45% (, respectively. Conclusion. The experimental membranes proved to regenerate bone around implants placed in fresh extraction sockets without soft tissue intrusion.

Carbon Molecular Sieve Membranes Derived from Tröger's Base-Based Microporous Polyimide for Gas Separation.

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Wang, Zhenggong; Ren, Huiting; Zhang, Shenxiang; Zhang, Feng; Jin, Jian

2018-03-09

Carbon molecular sieve (CMS)-based membranes have attracted great attention because of their outstanding gas-separation performance. The polymer precursor is a key point for the preparation of high-performance CMS membranes. In this work, a microporous polyimide precursor containing a Tröger's base unit was used for the first time to prepare CMS membranes. By optimizing the pyrolysis procedure and the soaking temperature, three TB-CMS membranes were obtained. Gas-permeation tests revealed that the comprehensive gas-separation performance of the TB-CMS membranes was greatly enhanced relative to that of most state-of-the-art CMS membranes derived from polyimides reported so far. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Derivation of the formula for the filtration coefficient by application of Poiseuille's law in membrane transport

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Maria Jarzyńska

2011-01-01

Full Text Available On the basis of Kedem-Katchalsky equations a mathematical analysis of volume flow (Jv of a binary solution through a membrane (M is presented. Two cases of transport generators have been considered: hydrostatic (Δp as well as osmotic (Δπ pressure difference. Based on the Poiseuille's law we derive the formula for the membrane filtration coefficient (Lp which takes into account the membrane properties, kinetic viscosity and density of a solution flowing across the membrane. With use of this formula we have made model calculations of the filtration coefficient Lp and volume flow Jv for a polymer membrane in the case when the solutions on both sides of the membrane are mixed.

Matrimid® derived carbon molecular sieve hollow fiber membranes for ethylene/ethane separation

KAUST Repository

Xu, Liren; Rungta, Meha; Koros, William J.

2011-01-01

materials in realistic gas separations. The very challenging ethylene/ethane separation is the primary target of this work. Matrimid® derived CMS hollow fiber membranes have been investigated in this work. Resultant CMS fiber showed interesting separation

Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells.

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Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

2016-09-30

Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

Hijacking of the Host Ubiquitin Network by Legionella pneumophila

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Jiazhang Qiu

2017-12-01

Full Text Available Protein ubiquitination is critical for regulation of numerous eukaryotic cellular processes such as protein homeostasis, cell cycle progression, immune response, DNA repair, and vesicular trafficking. Ubiquitination often leads to the alteration of protein stability, subcellular localization, or interaction with other proteins. Given the importance of ubiquitination in the regulation of host immunity, it is not surprising that many infectious agents have evolved strategies to interfere with the ubiquitination network with sophisticated mechanisms such as functional mimicry. The facultative intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila is phagocytosed by macrophages and is able to replicate within a niche called Legionella-containing vacuole (LCV. The biogenesis of LCV is dependent upon the Dot/Icm type IV secretion system which delivers more than 330 effector proteins into host cytosol. The optimal intracellular replication of L. pneumophila requires the host ubiquitin-proteasome system. Furthermore, membranes of the bacterial phagosome are enriched with ubiquitinated proteins in a way that requires its Dot/Icm type IV secretion system, suggesting the involvement of effectors in the manipulation of the host ubiquitination machinery. Here we summarize recent advances in our understanding of mechanisms exploited by L. pneumophila effector proteins to hijack the host ubiquitination pathway.

Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

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Schulz, Sarah; Wilkes, Martin; Mills, Deryck J; Kühlbrandt, Werner; Meier, Thomas

2017-04-01

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N 1 N o -type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c 17 ring is H + specific, demonstrating that the ATPase is proton-coupled. The c 17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

The Crucial Role of Biofilms in Cryptococcus neoformans Survival within Macrophages and Colonization of the Central Nervous System

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Lilit Aslanyan

2017-02-01

Full Text Available Cryptococcus neoformans is an encapsulated yeast-like fungus capable of causing life threatening meningoencephalitis in patients with impaired immunity. This microbe primarily infects the host via inhalation but has the ability to disseminate to the central nervous system (CNS either as a single cell or inside of macrophages. Upon traversing the blood brain barrier, C. neoformans has the capacity to form biofilm-like structures known as cryptococcomas. Hence, we will discuss the C. neoformans elements contributing to biofilm formation including the fungus’ ability to survive in the acidic environment of a macrophage phagosome and inside of the CNS. The purpose of this mini-review is to instill fresh interest in understanding the importance of biofilms on fungal pathogenesis.

The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy.

LENUS (Irish Health Repository)

Tangney, Mark

2012-01-31

Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.

Comparative anatomy of phagocytic and immunological synapses

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Florence eNiedergang

2016-01-01

Full Text Available The generation of phagocytic cups and immunological synapses are crucial events of the innate and adaptive immune responses, respectively. They are triggered by distinct immune receptors and performed by different cell types. However, growing experimental evidence shows that a very close series of molecular and cellular events control these two processes. Thus, the tight and dynamic interplay between receptor signaling, actin and microtubule cytoskeleton, and targeted vesicle traffic are all critical features to build functional phagosomes and immunological synapses. Interestingly, both phagocytic cups and immunological synapses display particular spatial and temporal patterns of receptors and signaling molecules, leading to the notion of phagocytic synapse. Here we discuss both types of structures, their organization and the mechanisms by which they are generated and regulated.

Membrane curvature stress and antibacterial activity of lactoferricin derivatives.

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Zweytick, Dagmar; Tumer, Sabine; Blondelle, Sylvie E; Lohner, Karl

2008-05-02

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.

Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

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Bernard Thay

Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane; Demangel, Caroline; Van Ingen, Jakko; Perez, Jorge; Baldeó n, Lucy R.; Abdallah, Abdallah; Caleechurn, Laxmee; Bottai, Daria; Van Zon, Maaike; De Punder, Karin; Van Der Laan, Tridia; Kant, Arie; Bossers-De Vries, Ruth; Willemsen, Peter Th J; Bitter, Wilbert M.; Van Soolingen, Dick; Brosch, Roland; Van Der Wel, Nicole N.; Peters, Peter J.

2012-01-01

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane

2012-05-08

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance

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Giang T. Nguyen

2017-08-01

Full Text Available Reactive oxygen species (ROS generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs, toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs. Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation

Human Amniotic Membrane-Derived Products in Sports Medicine: Basic Science, Early Results, and Potential Clinical Applications.

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Riboh, Jonathan C; Saltzman, Bryan M; Yanke, Adam B; Cole, Brian J

2016-09-01

Amniotic membrane (AM)-derived products have been successfully used in ophthalmology, plastic surgery, and wound care, but little is known about their potential applications in orthopaedic sports medicine. To provide an updated review of the basic science and preclinical and clinical data supporting the use of AM-derived products and to review their current applications in sports medicine. Systematic review. A systematic search of the literature was conducted using the Medline, EMBASE, and Cochrane databases. The search term amniotic membrane was used alone and in conjunction with stem cell, orthopaedic, tissue engineering, scaffold, and sports medicine. The search identified 6870 articles, 80 of which, after screening of the titles and abstracts, were considered relevant to this study. Fifty-five articles described the anatomy, basic science, and nonorthopaedic applications of AM-derived products. Twenty-five articles described preclinical and clinical trials of AM-derived products for orthopaedic sports medicine. Because the level of evidence obtained from this search was not adequate for systematic review or meta-analysis, a current concepts review on the anatomy, physiology, and clinical uses of AM-derived products is presented. Amniotic membranes have many promising applications in sports medicine. They are a source of pluripotent cells, highly organized collagen, antifibrotic and anti-inflammatory cytokines, immunomodulators, and matrix proteins. These properties may make it beneficial when applied as tissue engineering scaffolds, improving tissue organization in healing, and treatment of the arthritic joint. The current body of evidence in sports medicine is heavily biased toward in vitro and animal studies, with little to no human clinical data. Nonetheless, 14 companies or distributors offer commercial AM products. The preparation and formulation of these products alter their biological and mechanical properties, and a thorough understanding of these

Investigation of the Use of a Bio-Derived Solvent for Non-Solvent-Induced Phase Separation (NIPS Fabrication of Polysulfone Membranes

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Xiaobo Dong

2018-05-01

Full Text Available Organic solvents, such as N-methyl-2-pyrrolidone (NMP and dimethylacetamide (DMAc, have been traditionally used to fabricate polymeric membranes. These solvents may have a negative impact on the environment and human health; therefore, using renewable solvents derived from biomass is of great interest to make membrane fabrication sustainable. Methyl-5-(dimethylamino-2-methyl-5-oxopentanoate (Rhodiasolv PolarClean is a bio-derived, biodegradable, nonflammable and nonvolatile solvent. Polysulfone is a commonly used polymer to fabricate membranes due to its thermal stability, strong mechanical strength and good chemical resistance. From cloud point curves, PolarClean showed potential to be a solvent for polysulfone. Membranes prepared with PolarClean were investigated in terms of their morphology, porosity, water permeability and protein rejection, and were compared to membranes prepared with traditional solvents. The pores of polysulfone/PolarClean membranes were sponge-like, and the membranes displayed higher water flux values (176.0 ± 8.8 LMH along with slightly higher solute rejection (99.0 ± 0.51%. On the other hand, PSf/DMAc membrane pores were finger-like with lower water flux (63.1 ± 12.4 LMH and slightly lower solute rejection (96 ± 2.00% when compared to PSf/PolarClean membranes.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway [version 1; referees: 2 approved, 1 approved with reservations

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Paul Fineran

2016-11-01

Full Text Available Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC, a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway [version 2; referees: 2 approved, 2 approved with reservations

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Paul Fineran

2017-06-01

Full Text Available Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC, a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC.  Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed.  Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in

Influence of starvation, triton WR-1339 and [131I]-human serum albumin on rat liver lysosomes

International Nuclear Information System (INIS)

Harikumar, P.; Ninjoor, V.

1986-01-01

The response of rat liver lysosomes to starvation and administration of lysosomotropic agents viz. Triton WR-1339 and [ 131 I]-human serum albumin, was assessed in terms of their distribution pattern after isopycnic sucrose density gradient centrifugation. Starvation induced changes in lysosomes appeared to be similar to that produced by the detergent uptake. Both the treatments caused a distinct decline in the equilibration densities of the organelles. On the other hand, injected labelled protein failed to comigrate with the lysosomal markers in starved as well as Triton treated rats and conspicuously remained in a region of high specific gravity in the gradient. These findings indicate retarded fusion between secondary lysosomes and [ 131 I]-human serum albumin containing phagosomes in the livers of rats subjected to starvation or detergent treatment. (author)

Cloning and Expression of Listeria monocytogenes Listeriolysin O in Lactobacillus plantarum

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Masoumeh Hayati

2017-11-01

Full Text Available Background: The protein listeriolysin O (LLO encoded by hly gene, is one of the most important virulence factors of Listeria monocytogenes. This highly potent immunogenic cholesterol binding toxin has hemolytic activity, responsible for phagosomal membrane disruption and bacterial escape to the cytoplasm and facilitating the stimulation of CD8+ T cells and Th1 response. Recently pathobiotechnological vaccination using probiotic bacteria have been proposed. One of these strategies is expression of LLO in non-pathogenic bacteria such as lactic acid bacteria as delivery strains. Objectives: Our aim in this study was cloning of hly gene in a Lactobacillus species via pNZ8110, an inducible expression vector which is specific for Lactococcus species. Materials and Methods: hly gene was amplified by PCR and cloned into pNZ8110 by restriction enzymes cutting and ligation method. After transformation and propagation in E. coli MC1061 intermediate host, it was successfully electrotransformed into Lactobacillus plantarum. Results: Gel electrophoresis of colony PCR, extracted plasmids and restriction analysis along with sequencing confirmed the transformation. After induction using supernatant of nisin producer Lactococcus lactis NZ9700 strain, Expression of LLO was confirmed by SDS PAGE and western blot. Conclusion: Here, we have employed a nonpathogenic probiotic strain; Lactobacillus plantarum for the first time to express hly gene of Listeria monocytogenes in order to propose a new vaccine candidate.

Nanodiamond-chymotrypsin and nanodiamond-papain conjugates, their synthesis and activity and visualization of their interaction with cells using optical and electron microscopy.

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Golyshev, Sergey A; Berkovich, Anna K; Yakovlev, Ruslan Yu; Bystrov, Dmitry M; Ivanov, Nikita M; Balandina, Galina N; Rudenskaya, Galina N

2017-07-28

Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of the dNDs' surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding. Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved the preservation of the enzymes' composition during synthesis. The same assay was used to determine the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain. The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared to be approximately 40% of that of the native enzymes. The optimum pH values and stability under various conditions were determined. The sizes of resulting particles were measured using dynamic light scattering and direct electron microscopic observation. The enzyme conjugates were shown to be prone to aggregation, resulting in micrometer-sized particles. The ζ-potentials were measured and found to be positive for the conjugates. The conjugated enzymes were tested for biological activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules. Incubation with conjugated enzymes results in morphological changes of the cells but does not affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

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Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

Evaluation of a dental pulp-derived cell sheet cultured on amniotic membrane substrate.

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Honjo, Ken-ichi; Yamamoto, Toshiro; Adachi, Tetsuya; Amemiya, Takeshi; Mazda, Osam; Kanamura, Narisato; Kita, Masakazu

2015-01-01

Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.

PALLADIUM/COPPER ALLOY COMPOSITE MEMBRANES FOR HIGH TEMPERATURE HYDROGEN SEPARATION FROM COAL-DERIVED GAS STREAMS; F

International Nuclear Information System (INIS)

J. Douglas Way; Robert L. McCormick

2001-01-01

Recent advances have shown that Pd-Cu composite membranes are not susceptible to the mechanical, embrittlement, and poisoning problems that have prevented widespread industrial use of Pd for high temperature H(sub 2) separation. These membranes consist of a thin ((approx)10(micro)m) film of metal deposited on the inner surface of a porous metal or ceramic tube. Based on preliminary results, thin Pd(sub 60)Cu(sub 40) films are expected to exhibit hydrogen flux up to ten times larger than commercial polymer membranes for H(sub 2) separation, and resist poisoning by H(sub 2)S and other sulfur compounds typical of coal gas. Similar Pd-membranes have been operated at temperatures as high as 750 C. The overall objective of the proposed project is to demonstrate the feasibility of using sequential electroless plating to fabricate Pd(sub 60)Cu(sub 40) alloy membranes on porous supports for H(sub 2) separation. These following advantages of these membranes for processing of coal-derived gas will be demonstrated: High H(sub 2) flux; Sulfur tolerant, even at very high total sulfur levels (1000 ppm); Operation at temperatures well above 500 C; and Resistance to embrittlement and degradation by thermal cycling. The proposed research plan is designed to providing a fundamental understanding of: Factors important in membrane fabrication; Optimization of membrane structure and composition; Effect of temperature, pressure, and gas composition on H(sub 2) flux and membrane selectivity; and How this membrane technology can be integrated in coal gasification-fuel cell systems

Melanin targets LC3-associated phagocytosis (LAP): A novel pathogenetic mechanism in fungal disease.

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Chamilos, Georgios; Akoumianaki, Tonia; Kyrmizi, Irene; Brakhage, Axel; Beauvais, Anne; Latge, Jean-Paul

2016-05-03

Intracellular swelling of conidia of the major human airborne fungal pathogen Aspergillus fumigatus results in surface exposure of immunostimulatory pathogen-associated molecular patterns (PAMPs) and triggers activation of a specialized autophagy pathway called LC3-associated phagocytosis (LAP) to promote fungal killing. We have recently discovered that, apart from PAMPs exposure, cell wall melanin removal during germination of A. fumigatus is a prerequisite for activation of LAP. Importantly, melanin promotes fungal pathogenicity via targeting LAP, as a melanin-deficient A. fumigatus mutant restores its virulence upon conditional inactivation of Atg5 in hematopoietic cells of mice. Mechanistically, fungal cell wall melanin selectively excludes the CYBA/p22phox subunit of NADPH oxidase from the phagosome to inhibit LAP, without interfering with signaling regulating cytokine responses. Notably, inhibition of LAP is a general property of melanin pigments, a finding with broad physiological implications.

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

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Stefania ePizzimenti

2013-09-01

Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

Derivative expansion of one-loop effective energy of stiff membranes with tension

Science.gov (United States)

Borelli, M. E. S.; Kleinert, H.; Schakel, Adriaan M. J.

1999-03-01

With help of a derivative expansion, the one-loop corrections to the energy functional of a nearly flat, stiff membrane with tension due to thermal fluctuations are calculated in the Monge parametrization. Contrary to previous studies, an arbitrary tilt of the surface is allowed to exhibit the nontrivial relations between the different, highly nonlinear terms accompanying the ultraviolet divergences. These terms are shown to have precisely the same form as those in the original energy functional, as necessary for renormalizability. Also infrared divergences arise. These, however, are shown to cancel in a nontrivial way.

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

Science.gov (United States)

Nishida, Takashi; Hara, Naho; Watanabe, Kenta; Shimizu, Takashi; Fujishima, Masahiro; Watarai, Masahisa

2018-01-01

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires' disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena . The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila , but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC -deletion mutant. To assess the phagocytic activity of P. tetraurelia , Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella -containing vacuoles from P. tetraurelia . We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia

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Takashi Nishida

2018-04-01

Full Text Available Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308 and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae: Phagocytic Hemocytes in the Circulation and the Kidney.

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Juan A Cueto

Full Text Available Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy and transmission electron microscopy (TEM. Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules. Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets' occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently and may mean that

Salmonella Intracellular Lifestyles and Their Impact on Host-to-Host Transmission.

Science.gov (United States)

Pucciarelli, M Graciela; García-Del Portillo, Francisco

2017-07-01

More than a century ago, infections by Salmonella were already associated with foodborne enteric diseases with high morbidity in humans and cattle. Intestinal inflammation and diarrhea are hallmarks of infections caused by nontyphoidal Salmonella serovars, and these pathologies facilitate pathogen transmission to the environment. In those early times, physicians and microbiologists also realized that typhoid and paratyphoid fever caused by some Salmonella serovars could be transmitted by "carriers," individuals outwardly healthy or at most suffering from some minor chronic complaint. In his pioneering study of the nontyphoidal serovar Typhimurium in 1967, Takeuchi published the first images of intracellular bacteria enclosed by membrane-bound vacuoles in the initial stages of the intestinal epithelium penetration. These compartments, called Salmonella -containing vacuoles, are highly dynamic phagosomes with differing biogenesis depending on the host cell type. Single-cell studies involving real-time imaging and gene expression profiling, together with new approaches based on genetic reporters sensitive to growth rate, have uncovered unprecedented heterogeneous responses in intracellular bacteria. Subpopulations of intracellular bacteria displaying fast, reduced, or no growth, as well as cytosolic and intravacuolar bacteria, have been reported in both in vitro and in vivo infection models. Recent investigations, most of them focused on the serovar Typhimurium, point to the selection of persisting bacteria inside macrophages or following an autophagy attack in fibroblasts. Here, we discuss these heterogeneous intracellular lifestyles and speculate on how these disparate behaviors may impact host-to-host transmissibility of Salmonella serovars.

Deficiencies of the lipid-signaling enzymes phospholipase D1 and D2 alter cytoskeletal organization, macrophage phagocytosis, and cytokine-stimulated neutrophil recruitment.

Directory of Open Access Journals (Sweden)

Wahida H Ali

Full Text Available Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA, a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD, has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.

Innate immune recognition of Francisella tularensis: activation of type-I interferons and the inflammasome

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Jonathan Wiley Jones

2011-02-01

Full Text Available Francisella tularensis is an intracellular pathogen that can cause severe disease in a wide range of mammalian hosts. Primarily residing in host macrophages, F. tularensis escapes phagosomal degradation, and replicates in the macrophage cytosol. The macrophage uses a series of pattern recognition receptors to detect conserved microbial molecules from invading pathogens, and initiates an appropriate host response. In the cytosol, F. tularensis is recognized by the inflammasome, a multiprotein complex responsible for the activation of the cysteine protease caspase-1. Caspase-1 activation leads to processing and release of proinflammatory cytokines and host cell death. Here we review recent work on the molecular mechanisms of inflammasome activation by F. tularensis, and its consequences both in vitro and in vivo. Finally, we discuss the coordination between the inflammasome and other cytosolic host responses, and the evidence for F. tularensis virulence factors that suppress inflammasome activation.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

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Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

2015-08-18

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

Matrimid® derived carbon molecular sieve hollow fiber membranes for ethylene/ethane separation

KAUST Repository

Xu, Liren

2011-09-01

Carbon molecular sieve (CMS) membranes have shown promising separation performance compared to conventional polymeric membranes. Translating the very attractive separation properties from dense films to hollow fibers is important for applying CMS materials in realistic gas separations. The very challenging ethylene/ethane separation is the primary target of this work. Matrimid® derived CMS hollow fiber membranes have been investigated in this work. Resultant CMS fiber showed interesting separation performance for several gas pairs, especially high selectivity for C2H4/C2H6. Our comparative study between dense film and hollow fiber revealed very similar selectivity for both configurations; however, a significant difference exists in the effective separation layer thickness between precursor fibers and their resultant CMS fibers. SEM results showed that the deviation was essentially due to the collapse of the porous substructure of the precursor fiber. Polymer chain flexibility (relatively low glass transition temperature (Tg) for Matrimid® relative to actual CMS formation) appears to be the fundamental cause of substructure collapse. This collapse phenomenon must be addressed in all cases involving intense heat-treatment near or above Tg. We also found that the defect-free property of the precursor fiber was not a simple predictor of CMS fiber performance. Even some precursor fibers with Knudsen diffusion selectivity could be transformed into highly selective CMS fibers for the Matrimid® precursor. To overcome the permeance loss problem caused by substructure collapse, several engineering approaches were considered. Mixed gas permeation results under realistic conditions demonstrate the excellent performance of CMS hollow fiber membrane for the challenging ethylene/ethane separation. © 2011 Elsevier B.V.

MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

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Shelby, Shameka J; Colwill, Karen; Dhe-Paganon, Sirano; Pawson, Tony; Thompson, Debra A

2013-01-01

The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

Directory of Open Access Journals (Sweden)

Shameka J Shelby

Full Text Available The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE. A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999, purified and phosphorylated. Ni(2+-NTA pull downs were performed using 6xHis-rMERTK(571-999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α, VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS, siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

Phenotypic assays for Mycobacterium tuberculosis infection.

Science.gov (United States)

Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

2017-10-01

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite

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Marsh, M. E.; Ridall, A. L.; Azadi, P.; Duke, P. J.

2002-01-01

The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase.

The homeodomain derived peptide Penetratin induces curvature of fluid membrane domains.

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Antonin Lamazière

Full Text Available BACKGROUND: Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS: Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like membrane domains. CONCLUSIONS/SIGNIFICANCE: The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we

Bone Healing in Extraction Sockets Covered With Collagen Membrane Alone or Associated With Porcine-Derived Bone Graft: a Comparative Histological and Histomorphometric Analysis.

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Guarnieri, Renzo; Testarelli, Luca; Stefanelli, Luigi; De Angelis, Francesca; Mencio, Francesca; Pompa, Giorgio; Di Carlo, Stefano

2017-01-01

The present paper reports data of a randomized study aimed to analyse and compare the histologic and histomorphometric aspects of bone healing in extraction sites covered with collagen membrane alone or associated with porcine-derived bone graft. Thirty patients, with single extraction sockets without severe bone wall defects in the premolar/molar region, were included. Ten extraction sockets were grafted with porcine-derived bone and covered with collagen membrane (group 1), 10 sites were covered with collagen membrane alone (group 2), and 10 sites healed spontaneously (group 3). After 4 months of healing, 26 (8 in group 1, 9 in group 2, and 9 in group 3) bone core specimens were harvested for histologic evaluation, then dental implants were placed. Sites in the group 1 and in the group 2 showed similar histologic and histomorphometric results without significantly differences in the percentage of vital bone (57.43% [SD 4.8] vs. 60.01% [SD 3.2]), and non-mineralized connective tissue 22.99% (SD 5.3) vs. 18.53% (SD 6.2). In group 1 a 16.57% (SD 3.8) of residual material was found. Results showed that the use of collagen membrane alone or associated to porcine-derived bone improves the healing bone process compared to that of extraction sites spontaneously healed. Moreover, histomorphometric data related to bone quality, indicated that extraction sites without severe walls defects and with a vestibular bone thickness > 1.5 mm, treated with a low resorbtion rate collagen membrane alone, do not need more than 4 months for dental implant insertion.

Automated sampling and data processing derived from biomimetic membranes

DEFF Research Database (Denmark)

Perry, Mark; Vissing, Thomas; Boesen, P.

2009-01-01

data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition...... applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet (TM)) for efficient data management......Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new...

DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral model Aiptasia

KAUST Repository

Li, Yong

2017-11-03

The symbiotic relationship between cnidarians and dinoflagellates is the cornerstone of coral reef ecosystems. Although research is focusing on the molecular mechanisms underlying this symbiosis, the role of epigenetic mechanisms, which have been implicated in transcriptional regulation and acclimation to environmental change, is unknown. To assess the role of DNA methylation in the cnidarian-dinoflagellate symbiosis, we analyzed genome-wide CpG methylation, histone associations, and transcriptomic states of symbiotic and aposymbiotic anemones in the model system Aiptasia. We find methylated genes are marked by histone H3K36me3 and show significant reduction of spurious transcription and transcriptional noise, revealing a role of DNA methylation in the maintenance of transcriptional homeostasis. Changes in DNA methylation and expression show enrichment for symbiosis-related processes such as immunity, apoptosis, phagocytosis recognition and phagosome formation, and unveil intricate interactions between the underlying pathways. Our results demonstrate that DNA methylation provides an epigenetic mechanism of transcriptional homeostasis during symbiosis.

Efferocytosis is an innate antibacterial mechanism.

Science.gov (United States)

Martin, Constance J; Booty, Matthew G; Rosebrock, Tracy R; Nunes-Alves, Cláudio; Desjardins, Danielle M; Keren, Iris; Fortune, Sarah M; Remold, Heinz G; Behar, Samuel M

2012-09-13

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism. Copyright © 2012 Elsevier Inc. All rights reserved.

DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral model Aiptasia

KAUST Repository

Li, Yong; Liew, Yi Jin; Cui, Guoxin; Cziesielski, Maha J; Zahran, Noura Ibrahim Omar; Michell, Craig T; Voolstra, Christian R.; Aranda, Manuel

2017-01-01

The symbiotic relationship between cnidarians and dinoflagellates is the cornerstone of coral reef ecosystems. Although research is focusing on the molecular mechanisms underlying this symbiosis, the role of epigenetic mechanisms, which have been implicated in transcriptional regulation and acclimation to environmental change, is unknown. To assess the role of DNA methylation in the cnidarian-dinoflagellate symbiosis, we analyzed genome-wide CpG methylation, histone associations, and transcriptomic states of symbiotic and aposymbiotic anemones in the model system Aiptasia. We find methylated genes are marked by histone H3K36me3 and show significant reduction of spurious transcription and transcriptional noise, revealing a role of DNA methylation in the maintenance of transcriptional homeostasis. Changes in DNA methylation and expression show enrichment for symbiosis-related processes such as immunity, apoptosis, phagocytosis recognition and phagosome formation, and unveil intricate interactions between the underlying pathways. Our results demonstrate that DNA methylation provides an epigenetic mechanism of transcriptional homeostasis during symbiosis.

Phagocytosis in Teleosts. Implications of the New Cells Involved

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María Ángeles Esteban

2015-12-01

Full Text Available Phagocytosis is the process by which cells engulf some solid particles to form internal vesicles known as phagosomes. Phagocytosis is in fact a specific form of endocytosis involving the vesicular interiorization of particles. Phagocytosis is essentially a defensive reaction against infection and invasion of the body by foreign substances and, in the immune system, phagocytosis is a major mechanism used to remove pathogens and/or cell debris. For these reasons, phagocytosis in vertebrates has been recognized as a critical component of the innate and adaptive immune responses to pathogens. Furthermore, more recent studies have revealed that phagocytosis is also crucial for tissue homeostasis and remodeling. Professional phagocytes in teleosts are monocyte/macrophages, granulocytes and dendritic cells. Nevertheless, in recent years phagocytic properties have also been attributed to teleost lymphocytes and thrombocytes. The possible implications of such cells on this important biological process, new factors affecting phagocytosis, evasion of phagocytosis or new forms of phagocytosis will be considered and discussed.

Rhodococcus equi: the many facets of a pathogenic actinomycete.

Science.gov (United States)

Vázquez-Boland, José A; Giguère, Steeve; Hapeshi, Alexia; MacArthur, Iain; Anastasi, Elisa; Valero-Rello, Ana

2013-11-29

Rhodococcus equi is a soil-dwelling pathogenic actinomycete that causes pulmonary and extrapulmonary pyogranulomatous infections in a variety of animal species and people. Young foals are particularly susceptible and develop a life-threatening pneumonic disease that is endemic at many horse-breeding farms worldwide. R. equi is a facultative intracellular parasite of macrophages that replicates within a modified phagocytic vacuole. Its pathogenicity depends on a virulence plasmid that promotes intracellular survival by preventing phagosome-lysosome fusion. Species-specific tropism of R. equi for horses, pigs and cattle appears to be determined by host-adapted virulence plasmid types. Molecular epidemiological studies of these plasmids suggest that human R. equi infection is zoonotic. Analysis of the recently determined R. equi genome sequence has identified additional virulence determinants on the bacterial chromosome. This review summarizes our current understanding of the clinical aspects, biology, pathogenesis and immunity of this fascinating microbe with plasmid-governed infectivity. Copyright © 2013 Elsevier B.V. All rights reserved.

A role for NADPH oxidase in antigen presentation

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Gail J Gardiner

2013-09-01

Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells.

Directory of Open Access Journals (Sweden)

M Kristen Hall

Full Text Available The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these

Automated sampling and data processing derived from biomimetic membranes

International Nuclear Information System (INIS)

Perry, M; Vissing, T; Hansen, J S; Nielsen, C H; Boesen, T P; Emneus, J

2009-01-01

Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet(TM)) for efficient data management. The combined solution provides a cost efficient and fast way to acquire, process and administrate large amounts of voltage clamp data that may be too laborious and time consuming to handle manually. (communication)

Automated sampling and data processing derived from biomimetic membranes

Energy Technology Data Exchange (ETDEWEB)

Perry, M; Vissing, T; Hansen, J S; Nielsen, C H [Aquaporin A/S, Diplomvej 377, DK-2800 Kgs. Lyngby (Denmark); Boesen, T P [Xefion ApS, Kildegaardsvej 8C, DK-2900 Hellerup (Denmark); Emneus, J, E-mail: Claus.Nielsen@fysik.dtu.d [DTU Nanotech, Technical University of Denmark, DK-2800 Kgs. Lyngby (Denmark)

2009-12-15

Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet(TM)) for efficient data management. The combined solution provides a cost efficient and fast way to acquire, process and administrate large amounts of voltage clamp data that may be too laborious and time consuming to handle manually. (communication)

Membrane Disordering is not Sufficient for Membrane Permeabilization by Islet Amyloidogenic Polypeptide: Studies of IAPP(20-29) Fragments

Science.gov (United States)

Brender, Jeffrey R.; Heyl, Deborah L.; Samisetti, Shyamprasad; Kotler, Samuel A.; Osborne, Joshua M.; Pesaru, Ranadheer R.; Ramamoorthy, Ayyalusamy

2013-01-01

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that bind the membrane and cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected. PMID:23493863

A Salmonella typhimurium-translocated Glycerophospholipid:Cholesterol Acyltransferase Promotes Virulence by Binding to the RhoA Protein Switch Regions

Energy Technology Data Exchange (ETDEWEB)

LaRock, Doris L.; Brzovic, Peter S.; Levin, Itay; Blanc, Marie-Pierre; Miller, Samuel I.

2012-08-24

Salmonella enterica serovar typhimurium translocates a glycerophospholipid: cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.

Antimicrobial properties and membrane-active mechanism of a potential α-helical antimicrobial derived from cathelicidin PMAP-36.

Directory of Open Access Journals (Sweden)

Yinfeng Lv

Full Text Available Antimicrobial peptides (AMPs, which present in the non-specific immune system of organism, are amongst the most promising candidates for the development of novel antimicrobials. The modification of naturally occurring AMPs based on their residue composition and distribution is a simple and effective strategy for optimization of known AMPs. In this study, a series of truncated and residue-substituted derivatives of antimicrobial peptide PMAP-36 were designed and synthesized. The 24-residue truncated peptide, GI24, displayed antimicrobial activity comparable to the mother peptide PMAP-36 with MICs ranging from 1 to 4 µM, which is lower than the MICs of bee venom melittin. Although GI24 displayed high antimicrobial activity, its hemolytic activity was much lower than melittin, suggesting that GI24 have optimal cell selectivity. In addition, the crucial site of GI24 was identified through single site-mutation. An amino acid with high hydrophobicity at position 23 played an important role in guaranteeing the high antimicrobial activity of GI24. Then, lipid vesicles and whole bacteria were employed to investigate the membrane-active mechanisms. Membrane-simulating experiments showed that GI24 interacted strongly with negatively charged phospholipids and weakly with zwitterionic phospholipids, which corresponded well with the data of its biological activities. Membrane permeabilization and flow cytometry provide the evidence that GI24 killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. GI24 resulted in greater cell morphological changes and visible pores on cell membrane as determined using scanning electron microscopy (SEM and transmission electron microscope (TEM. Taken together, the peptide GI24 may provide a promising antimicrobial agent for therapeutic applications against the frequently-encountered bacteria.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence

Science.gov (United States)

Figueira, T. N.; Palermo, L. M.; Veiga, A. S.; Huey, D.; Alabi, C. A.; Santos, N. C.; Welsch, J. C.; Mathieu, C.; Niewiesk, S.; Moscona, A.

2016-01-01

ABSTRACT Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides. PMID:27733647

Amniotic membrane-derived stem cells: immunomodulatory properties and potential clinical application

Directory of Open Access Journals (Sweden)

Insausti CL

2014-03-01

Full Text Available Carmen L Insausti,1 Miguel Blanquer,1 Ana M García-Hernández,1 Gregorio Castellanos,2 José M Moraleda11Unidad de Trasplante Hematopoyético y Terapia Celular, 2Servicio de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, IMIB, Campus Mare Nostrum, Universidad de Murcia, El Palmar, Murcia, SpainAbstract: Epithelial and mesenchymal cells isolated from the amniotic membrane (AM possess stem cell characteristics, differentiation potential toward lineages of different germ layers, and immunomodulatory properties. While their expansion and differentiation potential have been well studied and characterized, knowledge about their immunomodulatory properties and the mechanisms involved is still incomplete. These mechanisms have been evaluated on various target cells of the innate and the adaptive system and in animal models of different inflammatory diseases. Some results have evidenced that the immunomodulatory effect of AM-derived cells is dependent on cell-cell contact, but many of them have demonstrated that these properties are mediated through the secretion of suppressive molecules. In this review, we present an update on the described immunomodulatory properties of the derived amniotic cells and some of the proposed involved mechanisms. Furthermore, we describe some assays in animal models of different inflammatory diseases which reveal the potential use of these cells to treat such diseases.Keywords: epithelial cells, mesenchymal cells, cell therapy, immunomodulation

Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

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Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

2017-06-01

Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Physicochemical characterization of artificial nanoerythrosomes derived from erythrocyte ghost membranes.

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Deák, Róbert; Mihály, Judith; Szigyártó, Imola Cs; Wacha, András; Lelkes, Gábor; Bóta, Attila

2015-11-01

Colloidal stabile nanoerythrosomes with 200 nm average diameter were formed from hemoglobin-free erythrocyte ghost membrane via sonication and membrane extrusion. The incorporation of extra lipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), added to the sonicated ghosts, caused significant changes in the thermotropic character of the original membranes. As a result of the increased DPPC ratio the chain melting of the hydrated DPPC system and the characteristic small angle X-ray scattering (SAXS) of the lipid bilayers appeared. Significant morphological changes were followed by transmission electron microscopy combined with freeze fracture method (FF-TEM). After the ultrasonic treatment the large entities of erythrocyte ghosts transformed into nearly spherical nanoerythrosomes with diameters between 100 and 300 nm and at the same time a great number of 10-30 nm large membrane proteins or protein clusters were dispersed in the aqueous medium. The infrared spectroscopy (FT-IR) pointed out, that the sonication did not cause changes in the secondary structures of the membrane proteins under our preparation conditions. About fivefold of extra lipid--compared to the lipid content of the original membrane--caused homogeneous dispersion of nanoerythrosomes however the shape of the vesicles was not uniform. After the addition of about tenfold of DPPC, monoform and monodisperse nanoerythrosomes became typical. The outer surfaces of these roughly spherical objects were frequently polygonal, consisting of a net of pentagons and hexagons. Copyright © 2015 Elsevier B.V. All rights reserved.

Pd Alloy Membranes for Hydrogen Separation from Coal-Derived Syngas

National Research Council Canada - National Science Library

Alptekin, Gokhan O; DeVoss, Sarah; Amalfitano, Bob; Way, Douglas; Thoen, Paul; Lusk, Mark

2006-01-01

TDA Research Inc., in collaboration with Colorado School of Mines (CSM) is developing a sulfur and CO-tolerant membrane to produce the clean hydrogen from syngas using Pd membrane films prepared on a variety of supports (e.g...

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

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Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

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Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Optimal operation of batch membrane processes

CERN Document Server

Paulen, Radoslav

2016-01-01

This study concentrates on a general optimization of a particular class of membrane separation processes: those involving batch diafiltration. Existing practices are explained and operational improvements based on optimal control theory are suggested. The first part of the book introduces the theory of membrane processes, optimal control and dynamic optimization. Separation problems are defined and mathematical models of batch membrane processes derived. The control theory focuses on problems of dynamic optimization from a chemical-engineering point of view. Analytical and numerical methods that can be exploited to treat problems of optimal control for membrane processes are described. The second part of the text builds on this theoretical basis to establish solutions for membrane models of increasing complexity. Each chapter starts with a derivation of optimal operation and continues with case studies exemplifying various aspects of the control problems under consideration. The authors work their way from th...

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

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Clancy, Danielle M; Sullivan, Graeme P; Moran, Hannah B T; Henry, Conor M; Reeves, Emer P; McElvaney, Noel G; Lavelle, Ed C; Martin, Seamus J

2018-03-13

Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Cathepsin B Improves ß-Amyloidosis and Learning and Memory in Models of Alzheimer's Disease.

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Embury, Christine M; Dyavarshetty, Bhagyalaxmi; Lu, Yaman; Wiederin, Jayme L; Ciborowski, Pawel; Gendelman, Howard E; Kiyota, Tomomi

2017-06-01

Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD), dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However, whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end, progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels, increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits.

Melanogenesis in dermal melanocytes of Japanese Silky chicken embryos.

Science.gov (United States)

Ortolani-Machado, C F; Freitas, P F; Faraco, C D

2009-08-01

The Japanese Silky chicken (SK) shows dermal and visceral hyperpigmentation. This study characterizes ultrastructurally the melanin granules developing in dermal melanocytes of the dorsal skin of SK, in an attempt to better understand the processes of melanogenesis in these permanently ectopic cells. The steps of melanogenesis are similar to those described for epidermal melanocytes, with melanosomes going from stage I to IV but, in SK, the maturation occurs in the cell body, as well as in the cytoplasmic processes. At stage III, the deposition of melanin is cumulative and can aggregate in rounded structures, which combine to turn into the mature granule. The final destiny of mature melanosomes is still unclear, although it was observed that dermal macrophages can accumulate melanin granules in their phagosomes. Even with the close proximity between melanocytes and other dermal cells, the transference of melanosomes was not observed. Our findings indicate that melanogenesis in dermal melanocytes in SK has the same morphological characteristics found in epidermal melanocytes, but the functional aspect still remains to be elucidated.

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence.

Science.gov (United States)

Hicks, Kevin G; Delbecq, Scott P; Sancho-Vaello, Enea; Blanc, Marie-Pierre; Dove, Katja K; Prost, Lynne R; Daley, Margaret E; Zeth, Kornelius; Klevit, Rachel E; Miller, Samuel I

2015-05-23

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Binding of two desmin derivatives to the plasma membrane and the nuclear envelope of avian erythrocytes: evidence for a conserved site-specificity in intermediate filament-membrane interactions

International Nuclear Information System (INIS)

Georgatos, S.D.; Weber, K.; Geisler, N.; Blobel, G.

1987-01-01

Using solution binding assays, the authors found that a 45-kDa fragment of 125 I-labelled desmin, lacking 67 residues from the N terminus, could specifically associate with avian erythrocyte nuclear envelopes but not with plasma membranes from the same cells. It was also observed that a 50-kDa desmin peptide, missing 27 C-terminal residues, retained the ability to bind to both membrane preparations. Displacement experiments with an excess of purified vimentin suggested that the two desmin derivatives were interacting with a previously identified vimentin receptor at the nuclear envelope, the protein lamin B. Additional analysis by affinity chromatography confirmed this conclusion. Employing an overlay assay, they demonstrated that the 50-kDa fragment, but not the 45-kDa desmin peptide, was capable of interacting with the plasma membrane polypeptide ankyrin (a known vimentin attachment site), as was intact vimentin. Conversely, the nuclear envelope protein lamin B was recognized by both fragments but not by a chymotryptic peptide composed solely of the helical rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helical rod domain, whereas the ankyrin-associating region is localized within its N-terminal head domain, exactly as in the case of vimentin

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

Science.gov (United States)

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

Science.gov (United States)

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Transcription factor σB plays an important role in the production of extracellular membrane-derived vesicles in Listeria monocytogenes.

Directory of Open Access Journals (Sweden)

Jung Hwa Lee

Full Text Available Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σ(B is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σ(B affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σ(B such as transporters (OpuCA and OpuCC, stress response (Kat, metabolism (LacD, translation (InfC, and cell division protein (FtsZ. Gene Ontology (GO enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σ(B plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

Modeling and simulation of anion-exchange membrane chromatography for purification of Sf9 insect cell-derived virus-like particles.

Science.gov (United States)

Ladd Effio, Christopher; Hahn, Tobias; Seiler, Julia; Oelmeier, Stefan A; Asen, Iris; Silberer, Christine; Villain, Louis; Hubbuch, Jürgen

2016-01-15

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of 'quality by design': High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Interactions at Membranes

DEFF Research Database (Denmark)

Jagalski, Vivien

. Today, we know more than ever before about the properties of biological membranes. Advanced biophysical techniques and sophisticated membrane models allow us to answer specific questions about the structure of the components within membranes and their interactions. However, many detailed structural...... the surface-immobilization of LeuT by exchanging the detergent with natural phosphatidylcholine (PC) lipids. Various surface sensitive techniques, including neutron reflectometry (NR), are employed and finally enabled us to confirm the gross structure of LeuT in a lipid environment as predicted by molecular...... dynamic simulations. In a second study, the co-localization of three toxic plant-derived diterpene resin acids (RAs) within DPPC membranes was investigated. These compounds are reported to disrupt the membrane and increase its fluidity. The RAs used in this study vary in their toxicity while...

Curvature recognition and force generation in phagocytosis

Directory of Open Access Journals (Sweden)

Prassler Jana

2010-12-01

Full Text Available Abstract Background The uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape. Results Using particles that consist of two lobes separated by a neck, we found that Dictyostelium cells transmit signals concerning the curvature of a surface to the actin system underlying the plasma membrane. Force applied to a concave region can divide a particle in two, allowing engulfment of the portion first encountered. The phagosome membrane that is bent around the concave region is marked by a protein containing an inverse Bin-Amphiphysin-Rvs (I-BAR domain in combination with an Src homology (SH3 domain, similar to mammalian insulin receptor tyrosine kinase substrate p53. Regulatory proteins enable the phagocyte to switch activities within seconds in response to particle shape. Ras, an inducer of actin polymerization, is activated along the cup surface. Coronin, which limits the lifetime of actin structures, is reversibly recruited to the cup, reflecting a program of actin depolymerization. The various forms of myosin-I are candidate motor proteins for force generation in particle uptake, whereas myosin-II is engaged only in retracting a phagocytic cup after a switch to particle release. Thus, the constriction of a phagocytic cup differs from the contraction of a cleavage furrow in mitosis. Conclusions Phagocytes scan a particle surface for convex and concave regions. By modulating the spatiotemporal pattern of actin organization, they are capable of switching between different modes of interaction with a particle, either arresting at a concave region and applying force in an attempt to sever the particle

Rheological properties of poly(vinyl alcohol) (PVA) derived composite membranes for fuel cells

Science.gov (United States)

Remiš, T.

2017-01-01

Rheological properties of new anhydrous proton conducting membrane based on PVA, tetraethyl orthosilicate (TEOS),sulfosuccinic acid (SSA), titanium dioxide (TiO2)was examined at various stoichiometric ratios. SSA was used as sulfonating agents to form a crosslinked structure and as proton source, whereas TEO Sand TiO2were utilized to improve the thermal and mechanical properties of the membrane. In order to verify that all the substances were immobilized into the matrix, the membranes were analysed by means of FT-IR. The rheological, mechanical and thermal properties of the membranes were investigated using rheometer ARES G2 and thermogravimetic analyser (TGA).The analysis of mixed PVA solutions exhibited a unique behaviour of viscosity with increased crosslink density. The dynamic storage modulus G´ of dried composite membranes shows better mechanical resistance and increased tolerance to pressure applied during membrane electrode assembly (MEA).

Critical composition fluctuations in artificial and cell-derived lipid membranes

Science.gov (United States)

Honerkamp-Smith, Aurelia

2014-03-01

Cell plasma membranes contain a mixture of lipid types which can segregate into coexisting liquids, a thermodynamic phenomenon which may contribute to biological functions. Simplified, artificial three-component lipid vesicles can be prepared which display a critical miscibility transition near room temperature. We found that such vesicles exhibit concentration fluctuations whose size, composition, and timescales vary consistently with critical exponents for two-dimensional conserved order parameter systems. However, the critical miscibility transition is also observed in vesicles formed directly from the membranes of living cells, despite their more complex composition and the presence of membrane proteins. I will describe our critical fluctuation measurements and also review a variety of more recent work by other researchers. Proximity to a critical point alters the spatial distribution and aggregation tendencies of proteins, and makes lipid mixtures more susceptible to domain formation by protein-mediated interactions, such as adhesion zones. Recent work suggests that critical temperature depression may also be relevant to the mechanism of anaesthetic action.

Effect of a bioactive curcumin derivative on DPPC membrane: A DSC and Raman spectroscopy study

Energy Technology Data Exchange (ETDEWEB)

Gardikis, Kostantinos [Department of Pharm. Technology, School of Pharmacy, University of Athens, Athens (Greece); Hatziantoniou, Sophia [Department of Pharm. Technology, School of Pharmacy, University of Athens, Athens (Greece); Viras, Kyriakos [Laboratory of Physical Chemistry, Department of Chemistry, University of Athens, Athens (Greece); Demetzos, Costas [Department of Pharm. Technology, School of Pharmacy, University of Athens, Athens (Greece)]. E-mail: demetzos@pharm.uoa.gr

2006-08-01

Interactions of dimethoxycurcumin (1) a lipophilic bioactive curcumin derivative with dipalmitoyl phosphatidylcholine (DPPC) were investigated. The thermodynamic changes caused by (1) and its location into DPPC lipid bilayers were monitored by differential scanning calorimetry and Raman spectroscopy. The results reveal that (1) influences the thermotropic properties of DPPC lipid membrane causing abolition of the pretransition and broadening of the phase-transition profile and slightly decreases the T {sub m} at increasing concentrations. The Raman height intensity ratios of the peaks I {sub 2935/2880}, I {sub 2844/2880} and I {sub 1090/1130} are representative of the interaction of (1) with the alkyl chains and furnish information about the ratio between disorder and order that exists in the conformation of the alkyl chain. The intensity changes of the peak at 715 cm{sup -1} indicates interaction between the choline head group and (1). The Raman spectroscopy results are in agreement with the thermal analysis results. Biologically active lipophilic molecules such as (1) should be studied in terms of their interaction with lipid bilayers prior to the development of advanced lipid carrier systems such as liposomes. The results of these studies provide information on the membrane integrity and physicochemical properties that are essential for the rational design lipidic drug delivery systems.

Rheological properties of poly(vinyl alcohol) (PVA) derived composite membranes for fuel cells

International Nuclear Information System (INIS)

Remiš, T

2017-01-01

Rheological properties of new anhydrous proton conducting membrane based on PVA, tetraethyl orthosilicate (TEOS),sulfosuccinic acid (SSA), titanium dioxide (TiO 2 )was examined at various stoichiometric ratios. SSA was used as sulfonating agents to form a crosslinked structure and as proton source, whereas TEO Sand TiO 2 were utilized to improve the thermal and mechanical properties of the membrane. In order to verify that all the substances were immobilized into the matrix, the membranes were analysed by means of FT-IR. The rheological, mechanical and thermal properties of the membranes were investigated using rheometer ARES G2 and thermogravimetic analyser (TGA).The analysis of mixed PVA solutions exhibited a unique behaviour of viscosity with increased crosslink density. The dynamic storage modulus G´ of dried composite membranes shows better mechanical resistance and increased tolerance to pressure applied during membrane electrode assembly (MEA). (paper)

Investigation of cobalt porphyrin doped polymer membrane films for the optical sensing of imidazole and its derivatives

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Yueyang Tan

2015-03-01

Full Text Available A cobalt(II porphyrin was successfully incorporated into polymer membranes for the optical sensing of imidazole and its derivatives. This research has led to a better understanding of the behavior of Co(II porphyrin in solution and in polymeric membranes. In aprotic dichloromethane (DCM, the Co(II tetraphenylporphyrin (CoTPP and Co(II octaethylporphyrin (CoOEP show a sensitive response to imidazole due to the strong ligation of the N-3 on the imidazole ring to the Co(II center, which induces an absorbance change to the Soret band. However, when doped in polymeric films, only the CoTPP exhibits moderate sensitivity towards aqueous imidazole, histamine and histidine. This weakened coordination ability of CoTPP towards imidazole in the polymer films may be due to the coordination of the plasticizer, the impurities from the THF and polymer matrix at the Co(II center. The selectivity of the polymer films towards imidazole over common anions is high. Lifetime of the cobalt(II porphyrin incorporated polymer film was relatively short.

Allogeneic amniotic membrane-derived mesenchymal stromal cell transplantation in a porcine model of chronic myocardial ischemia

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Kimura M

2012-01-01

Full Text Available Introduction. Amniotic membrane contains a multipotential stem cell population and is expected to possess the machinery to regulate immunological reactions. We investigated the safety and efficacy of allogeneic amniotic membrane-derived mesenchymal stromal cell (AMSC transplantation in a porcine model of chronic myocardial ischemia as a preclinical trial. Methods. Porcine AMSCs were isolated from amniotic membranes obtained by cesarean section just before delivery and were cultured to increase their numbers before transplantation. Chronic myocardial ischemia was induced by implantation of an ameroid constrictor around the left circumflex coronary artery. Four weeks after ischemia induction, nine swine were assigned to undergo either allogeneic AMSC transplantation or normal saline injection. Functional analysis was performed by echocardiography, and histological examinations were carried out by immunohistochemistry 4 weeks after AMSC transplantation. Results. Echocardiography demonstrated that left ventricular ejection fraction was significantly improved and left ventricular dilatation was well attenuated 4 weeks after AMSC transplantation. Histological assessment showed a significant reduction in percentage of fibrosis in the AMSC transplantation group. Injected allogeneic green fluorescent protein (GFP-expressing AMSCs were identified in the immunocompetent host heart without the use of any immunosuppressants 4 weeks after transplantation. Immunohistochemistry revealed that GFP colocalized with cardiac troponin T and cardiac troponin I. Conclusions. We have demonstrated that allogeneic AMSC transplantation produced histological and functional improvement in the impaired myocardium in a porcine model of chronic myocardial ischemia. The transplanted allogeneic AMSCs survived without the use of any immunosuppressants and gained cardiac phenotype through either their transdifferentiation or cell fusion.

Synthesis of Lipophilic Guanine N-9 Derivatives

DEFF Research Database (Denmark)

Wamberg, Michael C; Pedersen, Pernille L; Löffler, Philipp M G

2017-01-01

the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid...... bilayer structures, in which both alkyl chain length and attachment chemistry determined their interaction with the membrane. Incubation of these guanine conjugates, as solids, with a decanoic acid vesicle suspension, showed that ether- and triazole-linked C10 anchors yielded an increased partitioning...... of the guanine derivative into the membranous phase compared to directly N-9-linked saturated alkyl anchors. Decanoic acid vesicle membranes could be loaded with up to 5.5 mol % guanine derivative, a 6-fold increase over previous limits. Thus, anchor chemistries exhibiting favorable interactions with a bilayer...

Exclusive photorelease of signalling lipids at the plasma membrane.

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Nadler, André; Yushchenko, Dmytro A; Müller, Rainer; Stein, Frank; Feng, Suihan; Mulle, Christophe; Carta, Mario; Schultz, Carsten

2015-12-21

Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

Restored in vivo-like membrane lipidomics positively influence in vitro features of cultured mesenchymal stromal/stem cells derived from human placenta.

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Chatgilialoglu, Alexandros; Rossi, Martina; Alviano, Francesco; Poggi, Paola; Zannini, Chiara; Marchionni, Cosetta; Ricci, Francesca; Tazzari, Pier Luigi; Taglioli, Valentina; Calder, Philip C; Bonsi, Laura

2017-02-07

The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. Culturing hFM-MSCs alters their

Metabolism of periplasmic membrane-derived oligosaccharides by the predatory bacterium Bdellovibrio bacteriovorus 109J

International Nuclear Information System (INIS)

Ruby, E.G.; McCabe, J.B.

1988-01-01

Membrane-derived oligosaccharides (MDO), a class of osmotically active carbohydrates, are the major organic solutes present in the periplasm of Escherichia coli and many other gram-negative bacteria when cells are grown in a medium of low osmolarity. Analyses of growing cells of Bdellovibrio bacteriovorus, a gram-negative predator of other bacteria, have confirmed that they also synthesize a characteristic MDO-like class of oligosaccharides. The natural growth environment of bdellovibrios is the periplasm of other gram-negative bacteria. Because of this location, prey cell MDO constitute a potential source of organic nutrients for growing bdellovibrios. Using cells of E. coli whose MDO were 3 H labeled, we examined the extent to which B. bacteriovorus 109J metabolizes these prey cell components. Interestingly, there was neither significant degradation nor incorporation of prey cell MDO by bdellovibrios during the course of their intracellular growth. In fact, bdellovibrios had little capability either to degrade extracellular MDO that was made available to them or to transport glucose, the major monomeric constituent of prey cell MDO. Instead, periplasmic MDO were irreversibly lost to the extracellular environment during the period of bdellovibrio attack and penetration. Thus, although prey cell periplasmic proteins are retained, other important periplasmic components are released early in the bdellovibrio growth cycle. The loss of these MDO may aid in the destabilization of the prey cell plasma membrane, increasing the availability of cytoplasmic constituents to the periplasmic bdellovibrio

Novel adsorptive ultrafiltration membranes derived from polyvinyltetrazole-co-polyacrylonitrile for Cu(II) ions removal

KAUST Repository

Kumar, Mahendra; Shevate, Rahul; Hilke, Roland; Peinemann, Klaus-Viktor

2016-01-01

Novel adsorptive ultrafiltration membranes were manufactured from synthesized polyvinyltetrazole−co−polyacrylonitrile (PVT−co−PAN) by nonsolvent induced phase separation (NIPS). PVT−co−PAN with various degree of functionalization (DF) was synthesized via a [3+2] cycloaddition reaction at 60°C using a commercial PAN. PVT−co−PAN with varied DF was then explored to prepare adsorptive membranes. The membranes were characterized by surface zeta potential and static water contact angle measurements, scanning electron microscopy as well as atomic force microscopy (AFM) techniques. It was shown that PVT segments contributed to alter the pore size, charge and hydrophilic behavior of the membranes. The membranes became more negatively charged and hydrophilic after addition of PVT segments. The PVT segments in the membranes served as the major binding sites for adsorption of Cu(II) ions from aqueous solution. The maximum adsorption of Cu(II) ions by the membranes in static condition and in a continuous ultrafiltration of 10 ppm solution was attained at pH = 5. The adsorption data suggest that the Freundlich isotherm model describes well Cu(II) ions adsorption on the membranes from aqueous solution. The adsorption capacity obtained from the Freundlich isotherm model was 44.3 mg g−1; this value is higher than other membrane adsorption data reported in the literature. Overall, the membranes fabricated from PVT−co−PAN are attractive for efficient removal of heavy metal ions under the optimized conditions.

Novel adsorptive ultrafiltration membranes derived from polyvinyltetrazole-co-polyacrylonitrile for Cu(II) ions removal

KAUST Repository

Kumar, Mahendra

2016-05-04

Novel adsorptive ultrafiltration membranes were manufactured from synthesized polyvinyltetrazole−co−polyacrylonitrile (PVT−co−PAN) by nonsolvent induced phase separation (NIPS). PVT−co−PAN with various degree of functionalization (DF) was synthesized via a [3+2] cycloaddition reaction at 60°C using a commercial PAN. PVT−co−PAN with varied DF was then explored to prepare adsorptive membranes. The membranes were characterized by surface zeta potential and static water contact angle measurements, scanning electron microscopy as well as atomic force microscopy (AFM) techniques. It was shown that PVT segments contributed to alter the pore size, charge and hydrophilic behavior of the membranes. The membranes became more negatively charged and hydrophilic after addition of PVT segments. The PVT segments in the membranes served as the major binding sites for adsorption of Cu(II) ions from aqueous solution. The maximum adsorption of Cu(II) ions by the membranes in static condition and in a continuous ultrafiltration of 10 ppm solution was attained at pH = 5. The adsorption data suggest that the Freundlich isotherm model describes well Cu(II) ions adsorption on the membranes from aqueous solution. The adsorption capacity obtained from the Freundlich isotherm model was 44.3 mg g−1; this value is higher than other membrane adsorption data reported in the literature. Overall, the membranes fabricated from PVT−co−PAN are attractive for efficient removal of heavy metal ions under the optimized conditions.

A Pathogenic Potential of Acinetobacter baumannii-Derived Membrane Vesicles

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Jong Suk Jin

2011-12-01

Full Text Available Acinetobacter baumannii secretes outer membrane vesicles (OMVs. A. baumannii OMVs deliver many virulence factors to host cells and then induce cytotoxicity and innate immune response. OMVs secreted from bacteria contribute directly to host pathology during A. baumannii infection.

Comparison of exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells and synovial membrane-derived mesenchymal stem cells for the treatment of osteoarthritis.

Science.gov (United States)

Zhu, Yu; Wang, Yuchen; Zhao, Bizeng; Niu, Xin; Hu, Bin; Li, Qing; Zhang, Juntao; Ding, Jian; Chen, Yunfeng; Wang, Yang

2017-03-09

Osteoarthritis (OA) is the most common joint disease worldwide. In the past decade, mesenchymal stem cells (MSCs) have been used widely for the treatment of OA. A potential mechanism of MSC-based therapies has been attributed to the paracrine secretion of trophic factors, in which exosomes may play a major role. In this study, we aimed to compare the effectiveness of exosomes secreted by synovial membrane MSCs (SMMSC-Exos) and exosomes secreted by induced pluripotent stem cell-derived MSCs (iMSC-Exos) on the treatment of OA. Induced pluripotent stem cell-derived MSCs and synovial membrane MSCs were characterized by flow cytometry. iMSC-Exos and SMMSC-Exos were isolated using an ultrafiltration method. Tunable resistive pulse-sensing analysis, transmission electron microscopy, and western blots were used to identify exosomes. iMSC-Exos and SMMSC-Exos were injected intra-articularly in a mouse model of collagenase-induced OA and the efficacy of exosome injections was assessed by macroscopic, histological, and immunohistochemistry analysis. We also evaluated the effects of iMSC-Exos and SMMSC-Exos on proliferation and migration of human chondrocytes by cell-counting and scratch assays, respectively. The majority of iMSC-Exos and SMMSC-Exos were approximately 50-150 nm in diameter and expressed CD9, CD63, and TSG101. The injection of iMSC-Exos and SMMSC-Exos both attenuated OA in the mouse OA model, but iMSC-Exos had a superior therapeutic effect compared with SMMSC-Exos. Similarly, chondrocyte migration and proliferation were stimulated by both iMSC-Exos and SMMSC-Exos, with iMSC-Exos exerting a stronger effect. The present study demonstrated that iMSC-Exos have a greater therapeutic effect on OA than SMMSC-Exos. Because autologous iMSCs are theoretically inexhaustible, iMSC-Exos may represent a novel therapeutic approach for the treatment of OA.

Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study.

Science.gov (United States)

Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu

2014-01-01

Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds.

Preparation of hydrophilic and antifouling polysulfone ultrafiltration membrane derived from phenolphthalin by copolymerization method

International Nuclear Information System (INIS)

Liu, Zhixiao; Mi, Zhiming; Chen, Chunhai; Zhou, Hongwei; Zhao, Xiaogang; Wang, Daming

2017-01-01

Graphical abstract: The mechanisms fouling and cleaning process of PSF-COOH membranes (A) the content of carboxyl less than 80%. (B) the content of carboxyl at 80%, 100%. - Highlights: • Phenolphthalin (PPL) containing carboxyl was successfully introduced into the molecule backbone of polysulfone (PSF). • A series of PSF-COOH copolymers with different carboxylation degree was synthesized and prepared as ultrafiltration membranes. • The introduction of PPL significantly improved the hydrophilicity, permeation flux and antifouling property of membranes. • This method is valuable for large-scale industrial production of hydrophilic membrane material. - Abstract: In this task, carboxylated polysulfone (PSF-COOH) was achieved by introducing the monomer of phenolphthalin (PPL) containing carboxyl to the molecule backbone of polysulfone (PSF). And a series of PSF-COOH copolymers with different carboxylation degree was synthesized by adjusting the molar (%) of bisphenol A (BPA) and PPL in direct copolymerization method and was prepared as PSF-COOH ultrafiltration membranes via phase separation method. The effect of PPL molar (%) in copolymers on the morphology, hydrophilicity, permeation flux, antifouling and mechanical properties of membranes was investigated by scanning electron microscope (SEM), atomic force microscope (AFM), water contact angle, ultrafiltration experiments and universal testing machine, respectively. The results showed that with the increased carboxyl content in membranes, the hydrophilicity, permeation fluxes and antifouling properties of membranes gradually increased. When the molar (%) of PPL to BPA was 100:0, the membrane exhibited the highest pure water flux (329.6 L/m"2 h) and the maximum flux recovery rate (92.5%). When the content of carboxyl in the membrane was 80% or more, after three cycles of BSA solution (1 g/L) filtration, the flux recovery rate was basically constant or showed a slightly increase. Thus, it can achieve the goal of

Preparation of hydrophilic and antifouling polysulfone ultrafiltration membrane derived from phenolphthalin by copolymerization method

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhixiao; Mi, Zhiming; Chen, Chunhai; Zhou, Hongwei; Zhao, Xiaogang; Wang, Daming, E-mail: wangdaming@jlu.edu.cn

2017-04-15

Graphical abstract: The mechanisms fouling and cleaning process of PSF-COOH membranes (A) the content of carboxyl less than 80%. (B) the content of carboxyl at 80%, 100%. - Highlights: • Phenolphthalin (PPL) containing carboxyl was successfully introduced into the molecule backbone of polysulfone (PSF). • A series of PSF-COOH copolymers with different carboxylation degree was synthesized and prepared as ultrafiltration membranes. • The introduction of PPL significantly improved the hydrophilicity, permeation flux and antifouling property of membranes. • This method is valuable for large-scale industrial production of hydrophilic membrane material. - Abstract: In this task, carboxylated polysulfone (PSF-COOH) was achieved by introducing the monomer of phenolphthalin (PPL) containing carboxyl to the molecule backbone of polysulfone (PSF). And a series of PSF-COOH copolymers with different carboxylation degree was synthesized by adjusting the molar (%) of bisphenol A (BPA) and PPL in direct copolymerization method and was prepared as PSF-COOH ultrafiltration membranes via phase separation method. The effect of PPL molar (%) in copolymers on the morphology, hydrophilicity, permeation flux, antifouling and mechanical properties of membranes was investigated by scanning electron microscope (SEM), atomic force microscope (AFM), water contact angle, ultrafiltration experiments and universal testing machine, respectively. The results showed that with the increased carboxyl content in membranes, the hydrophilicity, permeation fluxes and antifouling properties of membranes gradually increased. When the molar (%) of PPL to BPA was 100:0, the membrane exhibited the highest pure water flux (329.6 L/m{sup 2} h) and the maximum flux recovery rate (92.5%). When the content of carboxyl in the membrane was 80% or more, after three cycles of BSA solution (1 g/L) filtration, the flux recovery rate was basically constant or showed a slightly increase. Thus, it can achieve the

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool.

Science.gov (United States)

Grabbe, Roman; Schmitz, Ruth A

2003-04-01

In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N2-fixation (nif) genes in response to molecular O2 and ammonium. We investigated the influence of membrane-bound oxidoreductases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH'-'lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in the presence of a redox-mediator or inside-out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif-induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif-induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen-limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif-induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD-moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen-limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD-cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL.

Novel tilt-curvature coupling in lipid membranes

Science.gov (United States)

Terzi, M. Mert; Deserno, Markus

2017-08-01

On mesoscopic scales, lipid membranes are well described by continuum theories whose main ingredients are the curvature of a membrane's reference surface and the tilt of its lipid constituents. In particular, Hamm and Kozlov [Eur. Phys. J. E 3, 323 (2000)] have shown how to systematically derive such a tilt-curvature Hamiltonian based on the elementary assumption of a thin fluid elastic sheet experiencing internal lateral pre-stress. Performing a dimensional reduction, they not only derive the basic form of the effective surface Hamiltonian but also express its emergent elastic couplings as trans-membrane moments of lower-level material parameters. In the present paper, we argue, though, that their derivation unfortunately missed a coupling term between curvature and tilt. This term arises because, as one moves along the membrane, the curvature-induced change of transverse distances contributes to the area strain—an effect that was believed to be small but nevertheless ends up contributing at the same (quadratic) order as all other terms in their Hamiltonian. We illustrate the consequences of this amendment by deriving the monolayer and bilayer Euler-Lagrange equations for the tilt, as well as the power spectra of shape, tilt, and director fluctuations. A particularly curious aspect of our new term is that its associated coupling constant is the second moment of the lipid monolayer's lateral stress profile—which within this framework is equal to the monolayer Gaussian curvature modulus, κ¯ m. On the one hand, this implies that many theoretical predictions now contain a parameter that is poorly known (because the Gauss-Bonnet theorem limits access to the integrated Gaussian curvature); on the other hand, the appearance of κ¯ m outside of its Gaussian curvature provenance opens opportunities for measuring it by more conventional means, for instance by monitoring a membrane's undulation spectrum at short scales.

Host Cell Responses to Persistent Mycoplasmas - Different Stages in Infection of HeLa Cells with Mycoplasma hominis

Science.gov (United States)

Hopfe, Miriam; Deenen, René; Degrandi, Daniel; Köhrer, Karl; Henrich, Birgit

2013-01-01

Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of

Crosslinked wholly aromatic polyether membranes based on quinoline derivatives and their application in high temperature polymer electrolyte membrane fuel cells

Science.gov (United States)

Kallitsis, K. J.; Nannou, R.; Andreopoulou, A. K.; Daletou, M. K.; Papaioannou, D.; Neophytides, S. G.; Kallitsis, J. K.

2018-03-01

An AB type difunctional quinoline based monomer bearing a pentafluorophenyl unit combined with a phenol functionality is being synthesized and homopolymerized to create linear aromatic polyethers as polymer electrolytes for HT-PEM FCs applications. Several conditions are tested for the optimized synthesis of the monomer and homopolymer. Additionally, covalent crosslinking through aromatic polyether bond formation enables the creation of wholly aromatic crosslinked polymeric electrolyte membranes. More specifically, the perfluorophenyl units are crosslinked with other hydroxyl end functionalized moieties, providing membranes with enhanced chemical and mechanical properties that are moreover easily doped with phosphoric acid even at ambient temperatures. All membranes are evaluated for their structural and thermal characteristics and their doping ability with phosphoric acid. Selected crosslinked membranes are further tested in terms of their single cell performance at the temperature range 160 °C-200 °C showing promising performance and high conductivity values even up to 0.2 S cm-1 in some cases.

A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

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Ulrike Resch

2016-11-01

Full Text Available Export of macromolecules via extracellular membrane-derived vesicles (MVs plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS, the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response.

Carbonate and Bicarbonate Ion Transport in Alkaline Anion Exchange Membranes

Science.gov (United States)

2013-06-25

comparable assumptions, a similar equation can be derived starting with the Nernst -Planck equation . σ = ∑ σi = ∑ F2z2i RT (ε− ε0)q D0i 1 + δi Ci [1] Using Eq...an appropriate ion-membrane diffusion coefficient. Finally, an equation derived from the dusty fluid model can be used to calculate the ionic...Finally, an equation derived from the dusty fluid model can be used to calculate the ionic conductivity of the membrane in different counter ion forms

The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells.

Science.gov (United States)

Arayan, Lauren Togonon; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

2015-06-01

This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

Science.gov (United States)

Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

2017-03-28

Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus -infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus -containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

Recognition of the mycobacterial cord factor by Mincle: relevance for granuloma formation and resistance to tuberculosis

Directory of Open Access Journals (Sweden)

Roland eLang

2013-01-01

Full Text Available The world’s most successful intracellular bacterial pathogen, Mycobacterium tuberculosis (MTB, survives inside macrophages by blocking phagosome maturation and establishes chronic infection characterized by the formation of granulomas. Trehalose-6,6-dimycolate (TDM, the mycobacterial cord factor, is the most abundant cell wall lipid of virulent mycobacteria, is sufficient to cause granuloma formation, and has long been known to be a major virulence factor of MTB. Recently, TDM has been shown to activate the Syk-Card9 signaling pathway in macrophages through binding to the C-type lectin receptor Mincle. The Mincle-Card9 pathway is required for activation of macrophages by TDM in vitro and for granuloma formation in vivo following injection of TDM. Whether this pathway is also exploited by MTB to reprogram the macrophage into a comfortable niche has not been explored yet. Several recent studies have investigated the phenotype of Mincle-deficient mice in mycobacterial infection, yielding divergent results in terms of a role for Mincle in host resistance. Here, we review these studies, discuss possible reasons for discrepant results and highlight open questions in the role of Mincle and other C-type lectin receptors in the infection biology of MTB.

The risk assessment of Gd_2O_3:Yb"3"+/Er"3"+ nanocomposites as dual-modal nanoprobes for magnetic and fluorescence imaging

International Nuclear Information System (INIS)

Huang, Long; Tian, Xiumei; Liu, Jun; Zheng, Cunjing; Xie, Fukang; Li, Li

2017-01-01

Our group has synthesized Gd_2O_3:Yb"3"+/Er"3"+ nanocomposites as magnetic/fluorescence imaging successfully in the previous study, which exhibit good uniformity and monodispersibility with a mean size of 7.4 nm. However, their systematic risk assessment remains unknown. In this article, the in vitro biocompatibility of the Gd_2O_3:Yb"3"+/Er"3"+ was assessed on the basis of cell viability and apoptosis. In vivo immunotoxicity was evaluated by monitoring the product of reactive oxygen species (ROS), clusters of differentiation (CD) markers, and superoxide dismutase (SOD) in Balb/c mice. No significant differences were found in cell viability, apoptosis, and immunotoxicity between our Gd_2O_3:Yb"3"+/Er"3"+ and gadodiamide which are used commonly in clinical. Few nanoprobes were localized in the phagosomes of the liver, heart, lung, spleen, kidney, brain, and tumor under the transmission electron microscopy (TEM) images. In addition, our products reveal good T_1-weighted contrast enhancement of xenografted murine tumor. Therefore, the above results may contribute to the effective application of Gd_2O_3:Yb"3"+/Er"3"+ as molecular imaging contrast agents and dual-modal nanoprobes for cancer detection.

Autophagic dedifferentiation induced by cooperation between TOR inhibitor and retinoic acid signals in budding tunicates.

Science.gov (United States)

Kawamura, Kaz; Yoshida, Takuto; Sekida, Satoko

2018-01-15

Asexual bud development in the budding tunicate Polyandrocarpa misakiensis involves transdifferentiation of multipotent epithelial cells, which is triggered by retinoic acid (RA), and thrives under starvation after bud isolation from the parent. This study aimed to determine cell and molecular mechanisms of dedifferentiation that occur during the early stage of transdifferentiation. During dedifferentiation, the numbers of autophagosomes, lysosomes, and secondary lysosomes increased remarkably. Mitochondrial degradation and exosome discharge also occurred in the atrial epithelium. Autophagy-related gene 7 (Atg7) and lysosomal proton pump A gene (PumpA) were activated during the dedifferentiation stage. When target of rapamycin (TOR) inhibitor was administered to growing buds without isolating them from the parent, phagosomes and secondary lysosomes became prominent. TOR inhibitor induced Atg7 only in the presence of RA. In contrast, when growing buds were treated with RA, lysosomes, secondary lysosomes, and mitochondrial degradation were prematurely induced. RA significantly activated PumpA in a retinoid X receptor-dependent manner. Our results indicate that in P. misakiensis, TOR inhibition and RA signals act in synergy to accomplish cytoplasmic clearance for dedifferentiation. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis and theory of gas transport in microporous sol-gel derived ceramic membranes

NARCIS (Netherlands)

de Lange, R.S.A.; de Lange, Rob; Keizer, Klaas; Burggraaf, Anthonie; Burggraaf, A.J.

1995-01-01

Sol-gel modification of mesoporous alumina membranes is a very successful technique to improve gas separation performance. Due to the formed microporous top layer, the membranes show activated transport and molecular sieve-like separation factors. This paper concentrates on the mechanism of

Specific ion effects on membrane potential and the permselectivity of ion exchange membranes.

Science.gov (United States)

Geise, Geoffrey M; Cassady, Harrison J; Paul, Donald R; Logan, Bruce E; Hickner, Michael A

2014-10-21

Membrane potential and permselectivity are critical parameters for a variety of electrochemically-driven separation and energy technologies. An electric potential is developed when a membrane separates electrolyte solutions of different concentrations, and a permselective membrane allows specific species to be transported while restricting the passage of other species. Ion exchange membranes are commonly used in applications that require advanced ionic electrolytes and span technologies such as alkaline batteries to ammonium bicarbonate reverse electrodialysis, but membranes are often only characterized in sodium chloride solutions. Our goal in this work was to better understand membrane behaviour in aqueous ammonium bicarbonate, which is of interest for closed-loop energy generation processes. Here we characterized the permselectivity of four commercial ion exchange membranes in aqueous solutions of sodium chloride, ammonium chloride, sodium bicarbonate, and ammonium bicarbonate. This stepwise approach, using four different ions in aqueous solution, was used to better understand how these specific ions affect ion transport in ion exchange membranes. Characterization of cation and anion exchange membrane permselectivity, using these ions, is discussed from the perspective of the difference in the physical chemistry of the hydrated ions, along with an accompanying re-derivation and examination of the basic equations that describe membrane potential. In general, permselectivity was highest in sodium chloride and lowest in ammonium bicarbonate solutions, and the nature of both the counter- and co-ions appeared to influence measured permselectivity. The counter-ion type influences the binding affinity between counter-ions and polymer fixed charge groups, and higher binding affinity between fixed charge sites and counter-ions within the membrane decreases the effective membrane charge density. As a result permselectivity decreases. The charge density and polarizability

Ion beam heating of thin silicon membranes

International Nuclear Information System (INIS)

Tissot, P.E.; Hart, R.R.

1993-01-01

For silicon membranes irradiated by an ion beam in a vacuum environment, such as the masks used for ion beam lithography and the membranes used for thin film self-annealing, the heat transfer modes are radiation and limited conduction through the thin membrane. The radiation component depends on the total hemispherical emissivity which varies with the thickness and temperature of the membrane. A semiempirical correlation for the absorption coefficient of high resistivity silicon was derived and the variation of the total emissivity with temperature was computed for membranes with thicknesses between 0.1 and 10 μm. Based on this result, the temperatures reached during exposure to ion beams of varying intensities were computed. A proper modeling of the emissivity is shown to be important for beam heating of thin silicon membranes. (orig.)

The biological activities of (1,3)-(1,6)-{beta}-d-glucan and porous electrospun PLGA membranes containing {beta}-glucan in human dermal fibroblasts and adipose tissue-derived stem cells

Energy Technology Data Exchange (ETDEWEB)

Woo, Yeon I; Park, Bong Joo; Kim, Hye-Lee; Lee, Mi Hee; Kim, Jungsung; Park, Jong-Chul [Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yang, Young-Il [Department of Pathology, School of Medicine, Paik Institute for Clinical Research, Inje University, 633-165 Gae-dong, Busan-jin-gu, Busan 614-735 (Korea, Republic of); Kim, Jung Koo [Department of Biomedical Engineering, College of Biomedical Science and Engineering, Inje University, Kimhae 621-749 (Korea, Republic of); Tsubaki, Kazufumi [R and D division, Asahi Denka Co. Ltd, 7-2-35 Higashi-ogu, Arakawa-ku, Tokyo 116-8554 (Japan); Han, Dong-Wook, E-mail: parkjc@yuhs.a [Department of Nanomedical Engineering, College of Nanoscience and Nanotechnology, Pusan National University, geumjeong-gu, Busan 609-735 (Korea, Republic of)

2010-08-01

In this study, we investigated the possible roles of (1,3)-(1,6)-{beta}-d-glucan ({beta}-glucan) and porous electrospun poly-lactide-co-glycolide (PLGA) membranes containing {beta}-glucan for skin wound healing, especially their effect on adult human dermal fibroblast (aHDF) and adipose tissue-derived stem cell (ADSC) activation, proliferation, migration, collagen gel contraction and biological safety tests of the prepared membrane. This study demonstrated that {beta}-glucan and porous PLGA membranes containing {beta}-glucan have enhanced the cellular responses, proliferation and migration, of aHDFs and ADSCs and the result of a collagen gel contraction assay also revealed that collagen gels contract strongly after 4 h post-gelation incubation with {beta}-glucan. Furthermore, we confirmed that porous PLGA membranes containing {beta}-glucan are biologically safe for wound healing study. These results indicate that the porous PLGA membranes containing {beta}-glucan interacted favorably with the membrane and the topical administration of {beta}-glucan was useful in promoting wound healing. Therefore, our study suggests that {beta}-glucan and porous PLGA membranes containing {beta}-glucan may be useful as a material for enhancing wound healing.

Cross flow microfiltration of oil-water emulsions using clay based ceramic membrane support and TiO2 composite membrane

OpenAIRE

Kanchapogu Suresh; G. Pugazhenthi

2017-01-01

The main objective of this work is to study the effect of cross flow filtration conditions on the separation of oily wastewater using ceramic support and TiO2 membrane. Firstly, the low cost clay based ceramic membrane support was prepared by uniaxial compaction method using combination of pyrophyllite, quartz, feldspar, kaolin, ball clay and calcium carbonate along with PVA as a binder. Subsequently, TiO2 composite membrane was fabricated via hydrothermal route employing TiO2 sol derived fro...

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

Science.gov (United States)

Bilyy, Rostyslav O; Shkandina, Tanya; Tomin, Andriy; Muñoz, Luis E; Franz, Sandra; Antonyuk, Volodymyr; Kit, Yuriy Ya; Zirngibl, Matthias; Fürnrohr, Barbara G; Janko, Christina; Lauber, Kirsten; Schiller, Martin; Schett, Georg; Stoika, Rostyslav S; Herrmann, Martin

2012-01-02

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Synthesis of Nanogels via Cell Membrane-Templated Polymerization.

Science.gov (United States)

Zhang, Jianhua; Gao, Weiwei; Fang, Ronnie H; Dong, Anjie; Zhang, Liangfang

2015-09-09

The synthesis of biomimetic hydrogel nanoparticles coated with a natural cell membrane is described. Compared to the existing strategy of wrapping cell membranes onto pre-formed nanoparticle substrates, this new approach forms the cell membrane-derived vesicles first, followed by growing nanoparticle cores in situ. It adds significant controllability over the nanoparticle properties and opens unique opportunities for a broad range of biomedical applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane Transporters as Mediators of Cisplatin Effects and Side Effects

Directory of Open Access Journals (Sweden)

Giuliano Ciarimboli

2012-01-01

Full Text Available Transporters are important mediators of specific cellular uptake and thus, not only for effects, but also for side effects, metabolism, and excretion of many drugs such as cisplatin. Cisplatin is a potent cytostatic drug, whose use is limited by its severe acute and chronic nephro-, oto-, and peripheral neurotoxicity. For this reason, other platinum derivatives, such as carboplatin and oxaliplatin, with less toxicity but still with antitumoral action have been developed. Several transporters, which are expressed on the cell membranes, have been associated with cisplatin transport across the plasma membrane and across the cell: the copper transporter 1 (Ctr1, the copper transporter 2 (Ctr2, the P-type copper-transporting ATPases ATP7A and ATP7B, the organic cation transporter 2 (OCT2, and the multidrug extrusion transporter 1 (MATE1. Some of these transporters are also able to accept other platinum derivatives as substrate. Since membrane transporters display a specific tissue distribution, they can be important molecules that mediate the entry of platinum derivatives in target and also nontarget cells possibly mediating specific effects and side effects of the chemotherapeutic drug. This paper summarizes the literature on toxicities of cisplatin compared to that of carboplatin and oxaliplatin and the interaction of these platinum derivatives with membrane transporters.

OXYGEN TRANSPORT CERAMIC MEMBRANES

International Nuclear Information System (INIS)

Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

2001-01-01

Conversion of natural gas to liquid fuels and chemicals is a major goal for the Nation as it enters the 21st Century. Technically robust and economically viable processes are needed to capture the value of the vast reserves of natural gas on Alaska's North Slope, and wean the Nation from dependence on foreign petroleum sources. Technologies that are emerging to fulfill this need are all based syngas as an intermediate. Syngas (a mixture of hydrogen and carbon monoxide) is a fundamental building block from which chemicals and fuels can be derived. Lower cost syngas translates directly into more cost-competitive fuels and chemicals. The currently practiced commercial technology for making syngas is either steam methane reforming (SMR) or a two-step process involving cryogenic oxygen separation followed by natural gas partial oxidation (POX). These high-energy, capital-intensive processes do not always produce syngas at a cost that makes its derivatives competitive with current petroleum-based fuels and chemicals. This project has the following 6 main tasks: Task 1--Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints. Task 2--Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability. Task 3--Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres. Task 4--Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures. Task 5--Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability. Task 6--Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques

Incorporation of Graphene-Related Carbon Nanosheets in Membrane Fabrication for Water Treatment: A Review

Directory of Open Access Journals (Sweden)

Jenny Lawler

2016-12-01

Full Text Available The minimization of the trade-off between the flux and the selectivity of membranes is a key area that researchers are continually working to optimise, particularly in the area of fabrication of novel membranes. Flux versus selectivity issues apply in many industrial applications of membranes, for example the unwanted diffusion of methanol in fuel cells, retention of valuable proteins in downstream processing of biopharmaceuticals, rejection of organic matter and micro-organisms in water treatment, or salt permeation in desalination. The incorporation of nanosheets within membrane structures can potentially lead to enhancements in such properties as the antifouling ability, hydrophilicy and permeability of membranes, with concomitant improvements in the flux/selectivity balance. Graphene nanosheets and derivatives such as graphene oxide and reduced graphene oxide have been investigated for this purpose, for example inclusion of nanosheets within the active layer of Reverse Osmosis or Nanofiltration membranes or the blending of nanosheets as fillers within Ultrafiltration membranes. This review summarizes the incorporation of graphene derivatives into polymeric membranes for water treatment with a focus on a number of industrial applications, including desalination and pharmaceutical removal, where enhancement of productivity and reduction in fouling characteristics have been afforded by appropriate incorporation of graphene derived nanosheets during membrane fabrication.

A Membrane Model from Implicit Elasticity Theory

Science.gov (United States)

Freed, A. D.; Liao, J.; Einstein, D. R.

2014-01-01

A Fungean solid is derived for membranous materials as a body defined by isotropic response functions whose mathematical structure is that of a Hookean solid where the elastic constants are replaced by functions of state derived from an implicit, thermodynamic, internal-energy function. The theory utilizes Biot’s (1939) definitions for stress and strain that, in 1-dimension, are the stress/strain measures adopted by Fung (1967) when he postulated what is now known as Fung’s law. Our Fungean membrane model is parameterized against a biaxial data set acquired from a porcine pleural membrane subjected to three, sequential, proportional, planar extensions. These data support an isotropic/deviatoric split in the stress and strain-rate hypothesized by our theory. These data also demonstrate that the material response is highly non-linear but, otherwise, mechanically isotropic. These data are described reasonably well by our otherwise simple, four-parameter, material model. PMID:24282079

Membrane-membrane interactions in a lipid-containing bacteriophage system. Progress report, October 1, 1978-September 30, 1979

International Nuclear Information System (INIS)

Snipes, W.

1979-06-01

Progress is reported on research on two aspects of the life cycle of PM2, a lipid-containing bacteriophage. The first concerns the initial interaction of PM2 with the outer membrane of its host cell, Pseudomonas BAL-31. The second concerns the assembly of PM2 in infected cells and the structural features of hydrophobic membrane perturbers that inhibit PM2 assembly. Several other projects have been completed: distribution of PM2 receptors; effects of adamantance derivatives on PM2 production; hydrophobic membrane perturbers as antiviral and virucidal agents; hydrophobic photosensitizers; and other technique development

Radiation effects on bovine taste bud membranes

International Nuclear Information System (INIS)

Shatzman, A.R.; Mossman, K.L.

1982-01-01

In order to investigate the mechanisms of radiation-induced taste loss, the effects of radiation on preparations of enriched bovine taste bud membranes were studied. Taste buds containing circumvallate papilae, and surrounding control epithelial tissues devoid of taste buds, were obtained from steers and given radiation doses of 0-7000 cGy (rad). Tissue fractions were isolated into membrane-enriched and heterogeneous components using differential and sucrose gradient centrifugation of tissue homogenates. The yield of membranes, as measured by protein content in the buoyant membrane-enriched fractions, was reduced in quantity with increasing radiation dose. The relation between radiation dose and membrane quantity in membrane-enriched fractions could be fit by a simple exponential model with taste bud-derived membranes twice as radiosensitive as membranes from control epithelial tissue. Binding of sucrose, sodium, and acetate and fluoride stimulation of adenylate cyclase were nearly identical in both irradiated and nonirradiated intact membranes. Radiation had no effect on fractions of heterogeneous components. While it is not clear what changes are occurring in enriched taste cell membranes, damage to membranes may play an important role in the taste loss observed in patients following radiotherapy

Anion-exchange membranes derived from quaternized polysulfone and exfoliated layered double hydroxide for fuel cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Wan; Liang, Na; Peng, Pai; Qu, Rong; Chen, Dongzhi; Zhang, Hongwei, E-mail: hanqiujiang@163.com

2017-02-15

Layered double hydroxides (LDH) are prepared by controlling urea assisted homogeneous precipitation conditions. Morphology and crystallinity of LDHs are confirmed by X-ray diffraction and scanning electron microscope. After LDHs are incorporated into quaternized polysulfone membranes, transmission electron microscope is used to observe the exfoliated morphology of LDH sheets in the membranes. The properties of the nanocomposite membranes, including water uptake, swelling ratio, mechanical property and ionic conductivity are investigated. The nanocomposite membrane containing 5% LDH sheets shows more balanced performances, exhibiting an ionic conductivity of 2.36×10{sup −2} S cm{sup −1} at 60 °C. - Graphical abstract: Anion-exchange membrane based on quaternized polysulfone and exfoliated layered double hydroxide is optically transparent and has good ionic properties.

Biomimetic devices functionalized by membrane channel proteins

Science.gov (United States)

Schmidt, Jacob

2004-03-01

We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

Specific ion effects on membrane potential and the permselectivity of ion exchange membranes

KAUST Repository

Geise, Geoffrey M.

2014-08-26

© the Partner Organisations 2014. Membrane potential and permselectivity are critical parameters for a variety of electrochemically-driven separation and energy technologies. An electric potential is developed when a membrane separates electrolyte solutions of different concentrations, and a permselective membrane allows specific species to be transported while restricting the passage of other species. Ion exchange membranes are commonly used in applications that require advanced ionic electrolytes and span technologies such as alkaline batteries to ammonium bicarbonate reverse electrodialysis, but membranes are often only characterized in sodium chloride solutions. Our goal in this work was to better understand membrane behaviour in aqueous ammonium bicarbonate, which is of interest for closed-loop energy generation processes. Here we characterized the permselectivity of four commercial ion exchange membranes in aqueous solutions of sodium chloride, ammonium chloride, sodium bicarbonate, and ammonium bicarbonate. This stepwise approach, using four different ions in aqueous solution, was used to better understand how these specific ions affect ion transport in ion exchange membranes. Characterization of cation and anion exchange membrane permselectivity, using these ions, is discussed from the perspective of the difference in the physical chemistry of the hydrated ions, along with an accompanying re-derivation and examination of the basic equations that describe membrane potential. In general, permselectivity was highest in sodium chloride and lowest in ammonium bicarbonate solutions, and the nature of both the counter- and co-ions appeared to influence measured permselectivity. The counter-ion type influences the binding affinity between counter-ions and polymer fixed charge groups, and higher binding affinity between fixed charge sites and counter-ions within the membrane decreases the effective membrane charge density. As a result permselectivity decreases. The

Permeability testing of biomaterial membranes

Energy Technology Data Exchange (ETDEWEB)

Dreesmann, L; Hajosch, R; Nuernberger, J Vaz; Schlosshauer, B [NMI Natural and Medical Sciences Institute at University Tuebingen, Markwiesenstr. 55, D-72770 Reutlingen (Germany); Ahlers, M [GELITA AG, Gammelsbacher Str. 2, D-69412 Eberbach (Germany)], E-mail: schlosshauer@nmi.de

2008-09-01

The permeability characteristics of biomaterials are critical parameters for a variety of implants. To analyse the permeability of membranes made from crosslinked ultrathin gelatin membranes and the transmigration of cells across the membranes, we combined three technical approaches: (1) a two-chamber-based permeability assay, (2) cell culturing with cytochemical analysis and (3) biochemical enzyme electrophoresis (zymography). Based on the diffusion of a coloured marker molecule in conjunction with photometric quantification, permeability data for a gelatin membrane were determined in the presence or absence of gelatin degrading fibroblasts. Cytochemical evaluation after cryosectioning of the membranes was used to ascertain whether fibroblasts had infiltrated the membrane inside. Zymography was used to investigate the potential release of proteases from fibroblasts, which are known to degrade collagen derivatives such as gelatin. Our data show that the diffusion equilibrium of a low molecular weight dye across the selected gelatin membrane is approached after about 6-8 h. Fibroblasts increase the permeability due to cavity formation in the membrane inside without penetrating the membrane for an extended time period (>21 days in vitro). Zymography indicates that cavity formation is most likely due to the secretion of matrix metalloproteinases. In summary, the combination of the depicted methods promises to facilitate a more rational development of biomaterials, because it provides a rapid means of determining permeability characteristics and bridges the gap between descriptive methodology and the mechanistic understanding of permeability alterations due to biological degradation.

Permeability testing of biomaterial membranes

International Nuclear Information System (INIS)

Dreesmann, L; Hajosch, R; Nuernberger, J Vaz; Schlosshauer, B; Ahlers, M

2008-01-01

The permeability characteristics of biomaterials are critical parameters for a variety of implants. To analyse the permeability of membranes made from crosslinked ultrathin gelatin membranes and the transmigration of cells across the membranes, we combined three technical approaches: (1) a two-chamber-based permeability assay, (2) cell culturing with cytochemical analysis and (3) biochemical enzyme electrophoresis (zymography). Based on the diffusion of a coloured marker molecule in conjunction with photometric quantification, permeability data for a gelatin membrane were determined in the presence or absence of gelatin degrading fibroblasts. Cytochemical evaluation after cryosectioning of the membranes was used to ascertain whether fibroblasts had infiltrated the membrane inside. Zymography was used to investigate the potential release of proteases from fibroblasts, which are known to degrade collagen derivatives such as gelatin. Our data show that the diffusion equilibrium of a low molecular weight dye across the selected gelatin membrane is approached after about 6-8 h. Fibroblasts increase the permeability due to cavity formation in the membrane inside without penetrating the membrane for an extended time period (>21 days in vitro). Zymography indicates that cavity formation is most likely due to the secretion of matrix metalloproteinases. In summary, the combination of the depicted methods promises to facilitate a more rational development of biomaterials, because it provides a rapid means of determining permeability characteristics and bridges the gap between descriptive methodology and the mechanistic understanding of permeability alterations due to biological degradation

Amniotic membrane for burn trauma

International Nuclear Information System (INIS)

Jamaluddin Zainol; Hasim Mohammad

1999-01-01

Amniotic membranes are derived from human placentae at birth. They have two layers mainly the amniotic and the chorionic surfaces which are separated by a thin layer of connective tissues. The two layers are separated during procurement, the placenta and the chorionic side are discarded and the amnion membranes are then further processed. Amnion membranes are normally procured from placentae which are normally free of infections, i.e; the mothers are antenatally screened for sexually transmitted diseases or AlDs related diseases. Intrapartum the mother should not be having chorioamnionitis or jaundice. Sometimes the amniotic membranes are acquired from fresh elective caeserian sections. After processing, the amniotic membranes are packed in two layers of polypropylene and radiated with cobalt 60 at a dose of about 25 kGy. The amniotic membranes are clinically used to cover burn surfaces especially effective for superficial or partial thickness burns. The thin membranes adhered well to the trauma areas and peeled off automatically by the second week. No change of dressing were necessary during these times because of the close adherence, there were less chance of external contamination or infections of these wounds. Due to their flexibility they are very useful to cover difference contours of the human body for example the face, body, elbows or knees. However our experience revealed that amniotic membranes are not useful for third degree bums because the membranes dissolves by the enzymes present in the wounds

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

Directory of Open Access Journals (Sweden)

Dagmar Zweytick

Full Text Available Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

Science.gov (United States)

Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

Ultraviolet- and sunlight-induced lipid peroxidation in liposomal membrane

International Nuclear Information System (INIS)

Mandal, T.K.; Chatterjee, S.N.

1980-01-01

Ultraviolet radiation and sunlight caused lipid peroxidation in the liposomal membrane (as detected by measurement of the oxidation index, A 233 /A 215 , and the amount of malondialdehyde formed) and made the membrane leaky (as revealed by the release of the trapped chromate anions). The oxidation index and the formation of malondialdehyde increased linearly with increasing dose of radiation and depended significantly on the dose rate. The effects were smaller in liposomes derived from Vibrio cholerae phospholipid than in those derived from egg lecithin. The effects of the radiation dose and dose rate on hemolysis and peroxidation (MDA formation) of the erythrocyte membrane followed a similar pattern. A direct correlation between the percentage leakage of chromate (Y) and the oxidation index (X) of the liposomal system was obtained as Y = 236.5 x X

Novel silica membranes for high temperature gas separations

KAUST Repository

Bighane, Neha

2011-04-01

This article describes fabrication of novel silica membranes derived via controlled oxidative thermolysis of polydimethylsiloxane and their gas separation performance. The optimized protocol for fabrication of the silica membranes is described and pure gas separation performance in the temperature range 35-80°C is presented. It is observed that the membranes exhibit activated transport for small gas penetrants such as He, H 2 and CO 2. The membranes can withstand temperatures up to 350°C in air and may ultimately find use in H 2/CO 2 separations to improve efficiency in the water-gas shift reactor process. © 2011 Elsevier B.V.

Gas Separation Membranes Derived from High-Performance Immiscible Polymer Blends Compatibilized with Small Molecules.

Science.gov (United States)

Panapitiya, Nimanka P; Wijenayake, Sumudu N; Nguyen, Do D; Huang, Yu; Musselman, Inga H; Balkus, Kenneth J; Ferraris, John P

2015-08-26

An immiscible polymer blend comprised of high-performance copolyimide 6FDA-DAM:DABA(3:2) (6FDD) and polybenzimidazole (PBI) was compatibilized using 2-methylimidazole (2-MI), a commercially available small molecule. Membranes were fabricated from blends of 6FDD:PBI (50:50) with and without 2-MI for H2/CO2 separations. The membranes demonstrated a matrix-droplet type microstructure as evident with scanning electron microscopy (SEM) imaging where 6FDD is the dispersed phase and PBI is the continuous phase. In addition, membranes with 2-MI demonstrated a uniform microstructure as observed by smaller and more uniformly dispersed 6FDD domains in contrast to 6FDD:PBI (50:50) blend membranes without 2-MI. This compatibilization effect of 2-MI was attributed to interfacial localization of 2-MI that lowers the interfacial energy similar to a surfactant. Upon the incorporation of 2-MI, the H2/CO2 selectivity improved remarkably, compared to the pure blend, and surpassed the Robeson's upper bound. To our knowledge, this is the first report of the use of a small molecule to compatibilize a high-performance immiscible polymer blend. This approach could afford a novel class of membranes in which immiscible polymer blends can be compatibilized in an economical and convenient fashion.

In vitro evaluation of biocompatibility of uncoated thermally reduced graphene and carbon nanotube-loaded PVDF membranes with adult neural stem cell-derived neurons and glia

Directory of Open Access Journals (Sweden)

Çagla Defterali

2016-12-01

Full Text Available Graphene, graphene-based nanomaterials (GBNs and carbon nanotubes (CNTs are being investigated as potential substrates for the growth of neural cells. However, in most in vitro studies the cells were seeded on these materials coated with various proteins implying that the observed effects on the cells could not solely be attributed to the GBN and CNT properties. Here we studied the biocompatibility of uncoated thermally reduced graphene (TRG and poly-vinylidene fluoride (PVDF membranes loaded with multi walled CNTs (MWCNTs using neural stem cells (NSCs isolated from the adult mouse olfactory bulb (termed aOBSCs. When aOBSCs were induced to differentiate on coverslips treated with TRG or control materials (polyethyleneimine-PEI and polyornithine plus fibronectin-PLO/F in a serum-free medium, neurons, astrocytes, and oligodendrocytes were generated in all conditions, indicating that TRG permits the multi-lineage differentiation of aOBSCs. However, the total number of cells was reduced on both PEI and TRG. In a serum-containing medium, aOBSC-derived neurons and oligodendrocytes grown on TRG were more numerous than in controls; the neurons developed synaptic boutons and oligodendrocytes were more branched. In contrast, neurons growing on PVDF membranes had reduced neurite branching and on MWCNTs-loaded membranes, oligodendrocytes were lower in numbers than in controls. Overall, these findings indicate that uncoated TRG may be biocompatible with the generation, differentiation, and maturation of aOBSC-derived neurons and glial cells, implying a potential use for TRG to study functional neuronal networks.

Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

Science.gov (United States)

Niessen, Anna; Heyder, Petra; Krienke, Stefan; Blank, Norbert; Tykocinski, Lars-Oliver; Lorenz, Hanns-Martin; Schiller, Martin

2015-07-15

A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material. © 2015. Published by The Company of Biologists Ltd.

Kinetic models of controllable pore growth of anodic aluminum oxide membrane

Science.gov (United States)

Huang, Yan; Zeng, Hong-yan; Zhao, Ce; Qu, Ye-qing; Zhang, Pin

2012-06-01

An anodized Al2O3 (AAO) membrane with apertures about 72 nm in diameter was prepared by two-step anodic oxidation. The appearance and pore arrangement of the AAO membrane were characterized by energy dispersive x-ray spectroscopy and scanning electron microscopy. It was confirmed that the pores with high pore aspect ratio were parallel, well-ordered, and uniform. The kinetics of pores growth in the AAO membrane was derived, and the kinetic models showed that pores stopped developing when the pressure ( σ) trended to equal the surface tension at the end of anodic oxidation. During pore expansion, the effects of the oxalic acid concentration and expansion time on the pore size were investigated, and the kinetic behaviors were explained with two kinetic models derived in this study. They showed that the pore size increased with extended time ( r= G· t+ G'), but decreased with increased concentration ( r = - K·ln c- K') through the derived mathematic formula. Also, the values of G, G', K, and K' were derived from our experimental data.

Dual-Bioinspired Design for Constructing Membranes with Superhydrophobicity for Direct Contact Membrane Distillation.

Science.gov (United States)

Zhu, Zhigao; Liu, Yuanren; Hou, Haoqing; Shi, Wenxin; Qu, Fangshu; Cui, Fuyi; Wang, Wei

2018-03-06

Water flux and durability are the two critical parameters that are closely associated with the practical application of membrane distillation (MD). Herein, we report a facile approach to fabricate superhydrophobic polyimide nanofibrous membranes (PI NFMs) with hierarchical structures, interconnected pores, and high porosity, which was derived from the electrospinning, dual-bioinspired design, and fluorination processes. Bioinspired adhesive based on polydopamine /polyethylenimine (PDA/PEI) composite was first linked onto membrane substrates and then assembled lotus leaf hierarchical structure by binding the negatively charged silica nanoparticles (SiO 2 NPs) via electrostatic attraction. The resultant superhydrophobic PI NFMs exhibit a water contact angle of 152°, robust hot water resistance of 85 °C, and high water entry pressure of 42 kPa. Moreover, the membrane with omniphobicity presents high water flux over 31 L m -2 h -1 and high salts rejection of ∼100% as well as robust durability for treating high salinity wastewater containing typical low surface tension and dissolved contaminants (Δ T = 40 °C). Significantly, the novel dual-bioinspired method can be used as a universal tool to modify various materials with hierarchical structures, which is expected to provide more effective alternative membranes for MD and even for other selective wetting separation fields.

Natural membranes of Hevea brasiliensis latex as delivery system for Casearia sylvestris leaf components

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Flávio A. Carvalho

Full Text Available ABSTRACT Natural latex from Hevea brasiliensis (Wild. ex A.Juss Müll.Arg., Euphorbiaceae, showed angiogenic action and Casearia sylvestris Sw., Salicaceae, leaf derivatives presented anti-inflammatory and wound healing activities. Therefore, an association of these effects was interesting for wound healing applications. The aims of this study were the development of membranes of natural latex incorporated with C. sylvestris leaf derivatives (ethanolic extract, diterpene concentrated fraction and casearin J, their chemical and physical characterization, and the evaluation of in vitro skin permeation and retention of C. sylvestris bioactive secondary metabolites (diterpenes and phenolic compounds. The membranes were developed mixing hydroethanolic solutions of C. sylvestris derivatives with latex and drying them in a desiccator. They were characterized by infrared spectroscopy, scanning electron microscopy, water vapor permeability and mechanical resistance assays, demonstrating that all membranes were permeable, resistant and homogeneous in surfaces. The permeation and retention assays demonstrated dermal penetration of phenolic compounds for ethanolic extract membrane and of casearin-like clerodane diterpenes for all membranes, indicating that these membranes have great potential for therapeutical application as a topical system for C. sylvestris components releasing.

Role of latent membrane protein 1 in chronic active Epstein–Barr virus infection-derived T/NK-cell proliferation

International Nuclear Information System (INIS)

Ito, Takuto; Kawazu, Hidetaka; Murata, Takayuki; Iwata, Seiko; Arakawa, Saki; Sato, Yoshitaka; Kuzushima, Kiyotaka; Goshima, Fumi; Kimura, Hiroshi

2014-01-01

Epstein–Barr virus (EBV) predominantly infects B cells and causes B-cell lymphomas, such as Burkitt lymphoma and Hodgkin lymphoma. However, it also infects other types of cells, including T and natural killer (NK) cells, and causes disorders, such as chronic active EBV infection (CAEBV) and T/NK-cell lymphoma. The CAEBV is a lymphoproliferative disease with poor prognosis, where EBV-positive T or NK cells grow rapidly, although the molecular mechanisms that cause the cell expansion still remain to be elucidated. EBV-encoded latent membrane protein 1 (LMP1) is an oncogene that can transform some cell types, such as B cells and mouse fibroblasts, and thus may stimulate cell proliferation in CAEBV. Here, we examined the effect of LMP1 on EBV-negative cells using the cells conditionally expressing LMP1, and on CAEBV-derived EBV-positive cells by inhibiting the function of LMP1 using a dominant negative form of LMP1. We demonstrated that LMP1 was responsible for the increased cell proliferation in the cell lines derived from CAEBV, while LMP1 did not give any proliferative advantage to the EBV-negative cell line

Free Vibration Analysis of Rectangular Orthotropic Membranes in Large Deflection

Directory of Open Access Journals (Sweden)

Zheng Zhou-Lian

2009-01-01

Full Text Available This paper reviewed the research on the vibration of orthotropic membrane, which commonly applied in the membrane structural engineering. We applied the large deflection theory of membrane to derive the governing vibration equations of orthotropic membrane, solved it, and obtained the power series formula of nonlinear vibration frequency of rectangular membrane with four edges fixed. The paper gave the computational example and compared the two results from the large deflection theory and the small one, respectively. Results obtained from this paper provide some theoretical foundation for the measurement of pretension by frequency method; meanwhile, the results provide some theoretical foundation for the research of nonlinear vibration of membrane structures and the response solving of membrane structures under dynamic loads.

Polymer-derived microporous ceramics for membranes and sensors for high temperature hydrogen purification and sensing

Energy Technology Data Exchange (ETDEWEB)

Prasad, Ravi Mohan

2012-06-11

The growing interest in the use of hydrogen as main fuel has increased the need for pure hydrogen (H{sub 2}) production and purification. There are several by-products (CO, H{sub 2}O, CO{sub 2}) associated with the production of hydrogen which might damage the production rate. Therefore, separation of hydrogen from other gases is an important step in the hydrogen production process. If H{sub 2} can be selectively removed from the product side during hydrogen production in membrane reactors, then it would be possible to achieve complete CO conversion in a single-step under high temperature conditions. The main goal of the present work is the high temperature H{sub 2} purification and sensing by applying polymer-derived ceramics. To prove the concept, the microporous SiBCN, Si{sub 3}N{sub 4} and SiCN ceramic membranes have been synthesized by the polymer-pyrolysis route and their performance for the hydrogen separation have been evaluated in tubular membranes as well as in planar chemiresistors. The synthesis of amorphous SiBCN ceramics has been realized through pyrolysis of poly(organoborosilazanes) in argon. Multilayered amorphous SiBCN/{gamma}-Al{sub 2}O{sub 3}/{alpha}-Al{sub 2}O{sub 3} membranes with gradient porosity have been realized and assessed with respect to the thermal stability, pore-size distribution and H{sub 2}/CO permeance. N{sub 2}-adsorption measurement indicates micropores in the range of 0.68-0.73 nm for three-fold SiBCN/{gamma}-Al{sub 2}O{sub 3}/{alpha}-Al{sub 2}O{sub 3} membrane. SEM characterization of three-fold SiBCN/{gamma}-Al{sub 2}O{sub 3}/{alpha}-Al{sub 2}O{sub 3} membrane shows the thickness of SiBCN membrane layer is 2.8 {mu}m; gas permeance measurements of the membrane shows H{sub 2}/CO selectivity of about 10.5 and the H{sub 2} permeance of about 1.05 x 10{sup -8} mol m{sup -2}s{sup -1}Pa{sup -1}. The observed gas permeation properties point out that the transportation of gas molecules through the membrane is governed by both

[The influence of N-, S-containing chinasolone derivatives (NC-224) on the biochemical and physicochemical parameters of membrane endoplasmatic reticulum and nuclear chromatine fractions of rats liver cells in conditions of its injury by tetrachloromethane].

Science.gov (United States)

Gubs'kyî, Iu I; Goriushko, G G; Belenichev, I F; Kovalenko, S I; Litvinova, N V; Marchenko, O M; Kurapova, T M; Babenko, L P; Velychko, O M

2010-01-01

Using biochemical and physicochemical methods of investigation in vivo, the effect of the substance NC-224, N-, S-chinasolone-derivative, on the lipoperoxidation activity in rat liver endoplasmatic reticulum membranes and nuclear chromatin fractions under tetrachloromethane intoxication have been studied. It was shown that NC-224 has pronounced antioxidant activity which is the biochemical basis of the substance membrane- and genome-protective effects and its ability to restore physicochemical properties of the surface and hydrophobic zones of hepatocyte membranes and structural parameter nuclear chromatin fractions in the conditions of chemical liver injury.

The State of Water in Proton Conducting Membranes

Energy Technology Data Exchange (ETDEWEB)

Allcock, Harry R.; Benesi, Alan; Macdonald, Digby D.

2010-08-27

The research carried out under grant No. DE-FG02-07ER46371, "The State of Water in Proton Conducting Membranes", during the period June 1, 2008 - May 31, 2010 was comprised of three related parts. These are: 1. An examination of the state of water in classical proton conduction membranes with the use of deuterium T1 NMR spectroscopy (Allcock and Benesi groups). 2. A dielectric relaxation examination of the behavior of water in classical ionomer membranes (Macdonald program). 3. Attempts to synthesize new proton-conduction polymers and membranes derived from the polyphosphazene system. (Allcock program) All three are closely related, crucial aspects of the design and development of new and improved polymer electrolyte fuel cell membranes on which the future of fuel cell technology for portable applications depends.

Carrier-facilitated transport of Cd(II) through a supported liquid membrane containing thiacalix[4]arene derivatives as ionophore

International Nuclear Information System (INIS)

Zaghbani, Asma; Tayeb, Rafik; Dhahbi, Mahmoud

2009-01-01

The feasibility of a facilitated transport process of cadmium ions through a SLM system incorporating new extractant agents, thiacalix[4]arenes, was studied. These molecules have sulfur atoms instead of usual methylene bridges. The chemical modification of the upper or the lower rim provides a great variety of supra molecules having different complexation ability and different conformational behaviour. The efficiency of the transport across the inner membrane organic liquid phase is shown to depend on the chemical (affinity) and structural (conformational states possible) parameters of these complexing molecules. In this work, two different thiacalix[4]arenes were selected as effective ionophore for the treatment of liquid media loaded in Cd(II). The results show that these thiacalix[4]arenes derivative ensure facilitated transport of cadmium cations through supported liquid membranes. Especially, the non-substituted thiacalix[4]arene can be considered as an effective extractant agent. The incidence of several parameters on transport efficiency such as pH of both aqueous solutions and carrier concentration was studied. The permeation of the species is due to a proton potential gradient (the driving force of the process) existing between the two opposite sides of the SLM. The initial flux, J, is found to be equal to 6.7.10 -7 mol.m -2 .s -1 , under optimal experimental conditions.

LL-37-derived membrane-active FK-13 analogs possessing cell selectivity, anti-biofilm activity and synergy with chloramphenicol and anti-inflammatory activity.

Science.gov (United States)

Rajasekaran, Ganesan; Kim, Eun Young; Shin, Song Yub

2017-05-01

Although the human-derived antimicrobial peptide (AMP) LL-37 has potent antimicrobial and anti-inflammatory activities, its therapeutic application is limited by its low cell selectivity and high production cost due to its large size. To overcome these problems, we tried to develop novel LL-37-derived short α-helical AMPs with improved cell selectivity and without a significant loss of anti-inflammatory activity relative to that of parental LL-37. Using amino acid substitution, we designed and synthesized a series of FK13 analogs based on the sequence of the 13-meric short FK13 peptide (residues 17-29 of LL-37) that has been identified as the region responsible for the antimicrobial activity of LL-37. Among the designed FK13 analogs, FK-13-a1 and FK-13-a7 showed high cell selectivity and retained the anti-inflammatory activity. The therapeutic index (a measure of cell selectivity) of FK-13-a1 and FK-13-a7 was 6.3- and 2.3-fold that of parental LL-37, respectively. Furthermore, FK-13-a1 and FK-13-a7 displayed more potent antimicrobial activity against antibiotic-resistant bacteria including MRSA, MDRPA, and VREF, than did LL-37. In addition, FK-13-a1 and FK-13-a7 exhibited greater synergistic effects with chloramphenicol against MRSA and MDRPA and were more effective anti-biofilm agents against MDRPA than LL-37 was. Moreover, FK-13-a1 and FK-13-a7 maintained their activities in the presence of physiological salts and human serum. SYTOX green uptake, membrane depolarization and killing kinetics revealed that FK13-a1 and FK13-a7 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Taken together, our results suggest that FK13-a1 and FK13-a7 can be developed as novel antimicrobial/anti-inflammatory agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Covalent modification of serum transferrin with phospholipid and incorporation into liposomal membranes

DEFF Research Database (Denmark)

Afzelius, P; Demant, E J; Hansen, Gert Helge

1989-01-01

A method is described for incorporation of water-soluble proteins into liposomal membranes using covalent protein-phospholipid conjugates in detergent solution. A disulfide derivative of phosphatidylethanolamine containing a reactive N-hydroxysuccinimide ester group is synthesized, and the deriva......A method is described for incorporation of water-soluble proteins into liposomal membranes using covalent protein-phospholipid conjugates in detergent solution. A disulfide derivative of phosphatidylethanolamine containing a reactive N-hydroxysuccinimide ester group is synthesized...

Quantum charged rigid membrane

Energy Technology Data Exchange (ETDEWEB)

Cordero, Ruben [Departamento de Fisica, Escuela Superior de Fisica y Matematicas del I.P.N., Unidad Adolfo Lopez Mateos, Edificio 9, 07738 Mexico, D.F. (Mexico); Molgado, Alberto [Unidad Academica de Fisica, Universidad Autonoma de Zacatecas, Zacatecas Zac. (Mexico); Rojas, Efrain, E-mail: cordero@esfm.ipn.mx, E-mail: amolgado@fisica.uaz.edu.mx, E-mail: efrojas@uv.mx [Departamento de Fisica, Facultad de Fisica e Inteligencia Artificial, Universidad Veracruzana, 91000 Xalapa, Veracruz (Mexico)

2011-03-21

The early Dirac proposal to model the electron as a charged membrane is reviewed. A rigidity term, instead of the natural membrane tension, involving linearly the extrinsic curvature of the worldvolume swept out by the membrane is considered in the action modeling the bubble in the presence of an electromagnetic field. We set up this model as a genuine second-order derivative theory by considering a non-trivial boundary term which plays a relevant part in our formulation. The Lagrangian in question is linear in the bubble acceleration and by means of the Ostrogradski-Hamiltonian approach, we observed that the theory comprises the management of both first- and second-class constraints. We thus show that our second-order approach is robust allowing for a proper quantization. We found an effective quantum potential which permits us to compute bounded states for the system. We comment on the possibility of describing brane world universes by invoking this kind of second-order correction terms.

Quantum charged rigid membrane

International Nuclear Information System (INIS)

Cordero, Ruben; Molgado, Alberto; Rojas, Efrain

2011-01-01

The early Dirac proposal to model the electron as a charged membrane is reviewed. A rigidity term, instead of the natural membrane tension, involving linearly the extrinsic curvature of the worldvolume swept out by the membrane is considered in the action modeling the bubble in the presence of an electromagnetic field. We set up this model as a genuine second-order derivative theory by considering a non-trivial boundary term which plays a relevant part in our formulation. The Lagrangian in question is linear in the bubble acceleration and by means of the Ostrogradski-Hamiltonian approach, we observed that the theory comprises the management of both first- and second-class constraints. We thus show that our second-order approach is robust allowing for a proper quantization. We found an effective quantum potential which permits us to compute bounded states for the system. We comment on the possibility of describing brane world universes by invoking this kind of second-order correction terms.

Membrane microparticles and diseases.

Science.gov (United States)

Wu, Z-H; Ji, C-L; Li, H; Qiu, G-X; Gao, C-J; Weng, X-S

2013-09-01

Membrane microparticles (MPs) are plasma membrane-derived vesicles shed by various types of activated or apoptotic cells including platelets, monocytes, endothelial cells, red blood cells, and granulocytes. MPs are being increasingly recognized as important regulators of cell-to-cell interactions. Recent evidences suggest they may play important functions not only in homeostasis but also in the pathogenesis of a number of diseases such as vascular diseases, cancer, infectious diseases and diabetes mellitus. Accordingly, inhibiting the production of MPs may serve as a novel therapeutic strategy for these diseases. Here we review recent advances on the mechanism underlying the generation of MPs and the role of MPs in vascular diseases, cancer, diabetes, inflammation, and pathogen infection.

Glycerol-plasticised silk membranes made using formic acid are ductile, transparent and degradation-resistant.

Science.gov (United States)

Allardyce, Benjamin J; Rajkhowa, Rangam; Dilley, Rodney J; Redmond, Sharon L; Atlas, Marcus D; Wang, Xungai

2017-11-01

Regenerated silk fibroin membranes tend to be brittle when dry. The use of plasticisers such as glycerol improve membrane ductility, but, when combined with aqueous processing, can lead to a higher degradation rate than solvent-annealed membranes. This study investigated the use of formic acid as the solvent with glycerol to make deformable yet degradation-resistant silk membranes. Here we show that membranes cast using formic acid had low light scattering, with a diffuse transmittance of less than 5% over the visible wavelengths, significantly lower than the 20% transmittance of aqueous derived silk/glycerol membranes. They had 64% β-sheet content and lost just 30% of the initial silk weight over 6h when tested with an accelerated enzymatic degradation assay, in comparison the aqueous membranes completely degraded within this timeframe. The addition of glycerol also improved the maximum elongation of formic acid derived membranes from under 3% to over 100%. They also showed good cytocompatibility and supported the adhesion and migration of human tympanic membrane keratinocytes. Formic acid based, silk/glycerol membranes may be of great use in medical applications such as repair of tympanic membrane perforation or ocular applications where transparency and resistance to enzymatic degradation are important. Copyright © 2017 Elsevier B.V. All rights reserved.

Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

International Nuclear Information System (INIS)

Hu, Dong; Wu, Jing; Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan; Xiao, Jian; Hu, Fengyu; Yang, Yabo; Zhang, Rongbo

2015-01-01

The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy

Global Liver Proteome Analysis Using iTRAQ Reveals AMPK-mTOR-Autophagy Signaling Is Altered by Intrauterine Growth Restriction in Newborn Piglets.

Science.gov (United States)

Long, Baisheng; Yin, Cong; Fan, Qiwen; Yan, Guokai; Wang, Zhichang; Li, Xiuzhi; Chen, Changqing; Yang, Xingya; Liu, Lu; Zheng, Zilong; Shi, Min; Yan, Xianghua

2016-04-01

Intrauterine growth restriction (IUGR) impairs fetal growth and development, perturbs nutrient metabolism, and increases the risk of developing diseases in postnatal life. However, the underlying mechanisms by which IUGR affects fetal liver development and metabolism remain incompletely understood. Here, we applied a high-throughput proteomics approach and biochemical analysis to investigate the impact of IUGR on the liver of newborn piglets. As a result, we identified 78 differentially expressed proteins in the three biological replicates, including 31 significantly up-regulated proteins and 47 significantly down-regulated proteins. Among them, a majority of differentially expressed proteins were related to nutrient metabolism and mitochondrial function. Additionally, many significantly down-regulated proteins participated in the mTOR signaling pathway and the phagosome maturation signaling pathway. Further analysis suggested that glucose concentration and hepatic glycogen storage were both reduced in IUGR newborn piglets, which may contribute to AMPK activation and mTORC1 inhibition. However, AMPK activation and mTORC1 inhibition failed to induce autophagy in the liver of IUGR neonatal pigs. A possible reason is that PP2Ac, a potential candidate in autophagy regulation, is significantly down-regulated in the liver of IUGR newborn piglets. These findings may provide implications for preventing and treating IUGR in human beings and domestic animals.

Intracellular degradation of microspheres based on cross-linked dextran hydrogels or amphiphilic block copolymers: A comparative Raman microscopy study

Science.gov (United States)

van Manen, Henk-Jan; van Apeldoorn, Aart A; Verrijk, Ruud; van Blitterswijk, Clemens A; Otto, Cees

2007-01-01

Micro- and nanospheres composed of biodegradable polymers show promise as versatile devices for the controlled delivery of biopharmaceuticals. Whereas important properties such as drug release profiles, biocompatibility, and (bio)degradability have been determined for many types of biodegradable particles, information about particle degradation inside phagocytic cells is usually lacking. Here, we report the use of confocal Raman microscopy to obtain chemical information about cross-linked dextran hydrogel microspheres and amphiphilic poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) microspheres inside RAW 264.7 macrophage phagosomes. Using quantitative Raman microspectroscopy, we show that the dextran concentration inside phagocytosed dextran microspheres decreases with cell incubation time. In contrast to dextran microspheres, we did not observe PEGT/PBT microsphere degradation after 1 week of internalization by macrophages, confirming previous studies showing that dextran microsphere degradation proceeds faster than PEGT/PBT degradation. Raman microscopy further showed the conversion of macrophages to lipid-laden foam cells upon prolonged incubation with both types of microspheres, suggesting that a cellular inflammatory response is induced by these biomaterials in cell culture. Our results exemplify the power of Raman microscopy to characterize microsphere degradation in cells and offer exciting prospects for this technique as a noninvasive, label-free optical tool in biomaterials histology and tissue engineering. PMID:17722552

Molecular Chaperone Dysfunction in Neurodegenerative Diseases and Effects of Curcumin

Directory of Open Access Journals (Sweden)

Panchanan Maiti

2014-01-01

Full Text Available The intra- and extracellular accumulation of misfolded and aggregated amyloid proteins is a common feature in several neurodegenerative diseases, which is thought to play a major role in disease severity and progression. The principal machineries maintaining proteostasis are the ubiquitin proteasomal and lysosomal autophagy systems, where heat shock proteins play a crucial role. Many protein aggregates are degraded by the lysosomes, depending on aggregate size, peptide sequence, and degree of misfolding, while others are selectively tagged for removal by heat shock proteins and degraded by either the proteasome or phagosomes. These systems are compromised in different neurodegenerative diseases. Therefore, developing novel targets and classes of therapeutic drugs, which can reduce aggregates and maintain proteostasis in the brains of neurodegenerative models, is vital. Natural products that can modulate heat shock proteins/proteosomal pathway are considered promising for treating neurodegenerative diseases. Here we discuss the current knowledge on the role of HSPs in protein misfolding diseases and knowledge gained from animal models of Alzheimer’s disease, tauopathies, and Huntington’s diseases. Further, we discuss the emerging treatment regimens for these diseases using natural products, like curcumin, which can augment expression or function of heat shock proteins in the cell.

Engineering nanoparticle-coated bacteria as oral DNA vaccines for cancer immunotherapy.

Science.gov (United States)

Hu, Qinglian; Wu, Min; Fang, Chun; Cheng, Changyong; Zhao, Mengmeng; Fang, Weihuan; Chu, Paul K; Ping, Yuan; Tang, Guping

2015-04-08

Live attenuated bacteria are of increasing importance in biotechnology and medicine in the emerging field of cancer immunotherapy. Oral DNA vaccination mediated by live attenuated bacteria often suffers from low infection efficiency due to various biological barriers during the infection process. To this end, we herein report, for the first time, a new strategy to engineer cationic nanoparticle-coated bacterial vectors that can efficiently deliver oral DNA vaccine for efficacious cancer immunotherapy. By coating live attenuated bacteria with synthetic nanoparticles self-assembled from cationic polymers and plasmid DNA, the protective nanoparticle coating layer is able to facilitate bacteria to effectively escape phagosomes, significantly enhance the acid tolerance of bacteria in stomach and intestines, and greatly promote dissemination of bacteria into blood circulation after oral administration. Most importantly, oral delivery of DNA vaccines encoding autologous vascular endothelial growth factor receptor 2 (VEGFR2) by this hybrid vector showed remarkable T cell activation and cytokine production. Successful inhibition of tumor growth was also achieved by efficient oral delivery of VEGFR2 with nanoparticle-coated bacterial vectors due to angiogenesis suppression in the tumor vasculature and tumor necrosis. This proof-of-concept work demonstrates that coating live bacterial cells with synthetic nanoparticles represents a promising strategy to engineer efficient and versatile DNA vaccines for the era of immunotherapy.

The risk assessment of Gd{sub 2}O{sub 3}:Yb{sup 3+}/Er{sup 3+} nanocomposites as dual-modal nanoprobes for magnetic and fluorescence imaging

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Huang, Long [Sun Yat-sen University, Department of Histology and Embryology, Zhongshan School of Medicine (China); Tian, Xiumei [Guangzhou Medical University, Department of Biomedical Engineering (China); Liu, Jun [Guangdong Ocean University, School of Electronics and Information Technology (China); Zheng, Cunjing; Xie, Fukang, E-mail: frankxie2000@yahoo.com [Sun Yat-sen University, Department of Histology and Embryology, Zhongshan School of Medicine (China); Li, Li, E-mail: li2@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center (China)

2017-02-15

Our group has synthesized Gd{sub 2}O{sub 3}:Yb{sup 3+}/Er{sup 3+} nanocomposites as magnetic/fluorescence imaging successfully in the previous study, which exhibit good uniformity and monodispersibility with a mean size of 7.4 nm. However, their systematic risk assessment remains unknown. In this article, the in vitro biocompatibility of the Gd{sub 2}O{sub 3}:Yb{sup 3+}/Er{sup 3+} was assessed on the basis of cell viability and apoptosis. In vivo immunotoxicity was evaluated by monitoring the product of reactive oxygen species (ROS), clusters of differentiation (CD) markers, and superoxide dismutase (SOD) in Balb/c mice. No significant differences were found in cell viability, apoptosis, and immunotoxicity between our Gd{sub 2}O{sub 3}:Yb{sup 3+}/Er{sup 3+} and gadodiamide which are used commonly in clinical. Few nanoprobes were localized in the phagosomes of the liver, heart, lung, spleen, kidney, brain, and tumor under the transmission electron microscopy (TEM) images. In addition, our products reveal good T{sub 1}-weighted contrast enhancement of xenografted murine tumor. Therefore, the above results may contribute to the effective application of Gd{sub 2}O{sub 3}:Yb{sup 3+}/Er{sup 3+} as molecular imaging contrast agents and dual-modal nanoprobes for cancer detection.

IFNγ-producing CD4+ T lymphocytes: the double-edged swords in tuberculosis.

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Kumar, Pawan

2017-12-01

IFNγ-producing CD4 + T cells (IFNγ + CD4 + T cells) are the key orchestrators of protective immunity against Mycobacterium tuberculosis (Mtb). Primarily, these cells act by enabling Mtb-infected macrophages to enforce phagosome-lysosome fusion, produce reactive nitrogen intermediates (RNIs), and activate autophagy pathways. However, TB is a heterogeneous disease and a host of clinical and experimental findings has also implicated IFNγ + CD4 + T cells in TB pathogenesis. High frequency of IFNγ + CD4 + T cells is the most invariable feature of the active disease. Active TB patients mount a heightened IFNγ + CD4 + T cell response to mycobacterial antigens and demonstrate an IFNγ-inducible transcriptomic signature. IFNγ + CD4 + T cells have also been shown to mediate TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) observed in a subset of antiretroviral therapy (ART)-treated HIV- and Mtb-coinfected people. The pathological face of IFNγ + CD4 + T cells during mycobacterial infection is further uncovered by studies in the animal model of TB-IRIS and in Mtb-infected PD-1 -/- mice. This manuscript encompasses the evidence supporting the dual role of IFNγ + CD4 + T cells during Mtb infection and sheds light on immune mechanisms involved in protection versus pathogenesis.

Transcriptional Analysis Allows Genome Reannotation and Reveals that Cryptococcus gattii VGII Undergoes Nutrient Restriction during Infection

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Patrícia Aline Gröhs Ferrareze

2017-08-01

Full Text Available Cryptococcus gattii is a human and animal pathogen that infects healthy hosts and caused the Pacific Northwest outbreak of cryptococcosis. The inhalation of infectious propagules can lead to internalization of cryptococcal cells by alveolar macrophages, a niche in which C. gattii cells can survive and proliferate. Although the nutrient composition of macrophages is relatively unknown, the high induction of amino acid transporter genes inside the phagosome indicates a preference for amino acid uptake instead of synthesis. However, the presence of countable errors in the R265 genome annotation indicates significant inhibition of transcriptomic analysis in this hypervirulent strain. Thus, we analyzed RNA-Seq data from in vivo and in vitro cultures of C. gattii R265 to perform the reannotation of the genome. In addition, based on in vivo transcriptomic data, we identified highly expressed genes and pathways of amino acid metabolism that would enable C. gattii to survive and proliferate in vivo. Importantly, we identified high expression in three APC amino acid transporters as well as the GABA permease. The use of amino acids as carbon and nitrogen sources, releasing ammonium and generating carbohydrate metabolism intermediaries, also explains the high expression of components of several degradative pathways, since glucose starvation is an important host defense mechanism.

Immune Evasion Strategies of Pathogens in Macrophages: the Potential for Limiting Pathogen Transmission.

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Ren, Yuwei; Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Zhang, Shujun

2017-01-01

Preventing pathogen transmission to a new host is of major interest to the immunologist and could benefit from a detailed investigation of pathogen immune evasion strategies. The first line of defense against pathogen invasion is provided by macrophages. When they sense pathogens, macrophages initiate signals to inflammatory and pro-inflammatory cytokines through pattern recognition receptors (PRRs) subsequently mediating phagocytosis and inflammation. The macrophage immune machinery classically includes two subsets: the activated M1 and the activated M2 that respond accordingly in diverse immune challenges. The lipid and glycogen metabolic pathways work together with the lysosome to help the mature phagosome to degrade and eliminate intracellular pathogens in macrophages. The viral evasion strategies are even more complex due to the interplay between autophagy and apoptosis. However, pathogens evolve several strategies to camouflage themselves against immune responses in order to ensure their survival, replication and transmission. These strategies include the muting of PRRs initiated inflammatory responses, attenuation of M1 and/or induction of M2 macrophages, suppression of autophago-lysosomal formation, interference with lipid and glycogen metabolism, and viral mediation of autophagy and apoptosis cross-talk to enhance viral replication. This review focuses on pathogen immune evasion methods and on the strategies used by the host against camouflaged pathogens.

Expression of Nocardia brasiliensis superoxide dismutase during the early infection of murine peritoneal macrophages.

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Revol, Agnès; Espinoza-Ruiz, Marisol; Medina-Villanueva, Igor; Salinas-Carmona, Mario Cesar

2006-12-01

Nocardia brasiliensis is the main agent of actinomycetoma in Mexico, but little is known about its virulence and molecular pathogenic pathways. These facultative intracellular bacteria are able to survive and divide within the host phagocytic cells, in part by neutralizing the reactive oxygen intermediates. Superoxide dismutase (SOD) participates in the intracellular survival of several bacterial species and, in particular, constitutes one of Nocardia asteroides virulence factors. To clarify SOD participation in the N. brasiliensis early infective process, we report its isolation and the consequent comparison of its transcript level. A 630 bp polymerase chain reaction fragment that included most of the coding sequence of N. brasiliensis sodA was cloned. A competitive assay was developed, allowing comparison of bacterial sod expression in exponential culture and 1 h after infecting peritoneal macrophages from BALB/c mice. At that time, there were viable bacteria in the macrophages. The intracellular bacteria presented a clear decrease in their sod transcript amount, although their 16S rRNA (used as an internal control) and hsp levels were maintained or slightly increased, respectively. These results indicate that sodA transcription is not maintained within the SOS bacterial response induced by phagosomal conditions. Further kinetics will be necessary to precisely define sod transcriptional regulation during N. brasiliensis intra-macrophage growth.

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

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Elena Ufimtseva

2015-01-01

Full Text Available Tuberculosis (TB is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals.

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

Science.gov (United States)

2015-01-01

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals. PMID:26064970

Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

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Karen K Y Lam

Full Text Available Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

Science.gov (United States)

Lam, Karen K Y; Zheng, Xingji; Forestieri, Roberto; Balgi, Aruna D; Nodwell, Matt; Vollett, Sarah; Anderson, Hilary J; Andersen, Raymond J; Av-Gay, Yossef; Roberge, Michel

2012-01-01

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Irgm1-deficient mice exhibit Paneth cell abnormalities and increased susceptibility to acute intestinal inflammation.

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Liu, Bo; Gulati, Ajay S; Cantillana, Viviana; Henry, Stanley C; Schmidt, Elyse A; Daniell, Xiaoju; Grossniklaus, Emily; Schoenborn, Alexi A; Sartor, R Balfour; Taylor, Gregory A

2013-10-15

Crohn's disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the immunity-related GTPase (IRG) M (IRGM). IRGs comprise a family of proteins known to confer resistance to intracellular infections through various mechanisms, including regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. We investigated the involvement of Irgm1, an ortholog of IRGM, in the genesis of murine intestinal inflammation. After dextran sodium sulfate exposure, Irgm1-deficient [Irgm1 knockout (KO)] mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1 KO mice, even without dextran sodium sulfate exposure, and were associated with impaired mitophagy and autophagy in Irgm1 KO intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes, that may be pivotal for proper Paneth cell functioning.

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

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Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

2017-09-01

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

PLD$ is involved in phagocytosis of microglia: expression and localization changes of PLD4 are correlated with activation state of microglia.

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Yoshinori Otani

Full Text Available Phospholipase D4 (PLD4 is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6 showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.

Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

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Hu, Dong, E-mail: austhudong@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wu, Jing, E-mail: wujing8008@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Hu, Fengyu; Yang, Yabo [Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Rongbo, E-mail: lory456@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China)

2015-05-29

The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.

Scale up of proteoliposome derived Cochleate production.

Science.gov (United States)

Zayas, Caridad; Bracho, Gustavo; Lastre, Miriam; González, Domingo; Gil, Danay; Acevedo, Reinaldo; del Campo, Judith; Taboada, Carlos; Solís, Rosa L; Barberá, Ramón; Pérez, Oliver

2006-04-12

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Ion Transport through Diffusion Layer Controlled by Charge Mosaic Membrane

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Akira Yamauchi

2012-01-01

Full Text Available The kinetic transport behaviors in near interface of the membranes were studied using commercial anion and cation exchange membrane and charge mosaic membrane. Current-voltage curve gave the limiting current density that indicates the ceiling of conventional flux. From chronopotentiometry above the limiting current density, the transition time was estimated. The thickness of boundary layer was derived with conjunction with the conventional limiting current density and the transition time from steady state flux. On the other hand, the charge mosaic membrane was introduced in order to examine the ion transport on the membrane surface in detail. The concentration profile was discussed by the kinetic transport number with regard to the water dissociation (splitting on the membrane surface.

Wastewater treatment with ion-exchange chitin membrane

International Nuclear Information System (INIS)

Paulenova, A.; Fjeld, R. A.; Visacky, V.

2001-01-01

Chitin, poly(N-acetyl-D glucosamine) and chitosan, its deacetylated derivates have recently obtained attention as bio-sorbents, because they shown a great ability to accumulate heavy metals and other pollutants. It was found that recovery of metals is strongly affected by pH. At low acidic pH range 4-5 chitin membrane exhibits better selectivity for lead than for cadmium or zinc. Sorption preference for metals decreases in the order: Pb > Cd > Zn. For uranium, as well for strontium was observed significant increase of recovery at decrease of pH to slightly acidic, close to neutral value. It was shown that chemical behavior of chitin membrane is excellent; ion-exchange nature of chitin was not changed during chitin membrane manufacturing process. Using of chitin membrane instead of chitin flake column brings significant increasing of driving force of the separation process, limited in the case of column experimental design by diffusion coefficient, while in the case of membrane process only by mass transfer coefficient. (authors)

Olefins-selective asymmetric carbon molecular sieve hollow fiber membranes for hybrid membrane-distillation processes for olefin/paraffin separations

KAUST Repository

Xu, Liren

2012-12-01

In this paper, the development of asymmetric carbon molecular sieve (CMS) hollow fiber membranes and advanced processes for olefin/paraffin separations based on the CMS membranes are reported. Membrane-based olefin/paraffin separations have been pursued extensively over the past decades. CMS membranes are promising to exceed the performance upper bound of polymer materials and have demonstrated excellent stability for gas separations. Previously, a substructure collapse phenomenon was found in Matrimid ® precursor derived CMS fiber. To overcome the permeance loss due to the increased separation layer thickness, 6FDA-DAM and 6FDA/BPDA-DAM precursors were selected as potential new precursors for carbon membrane formation. Defect-free asymmetric 6FDA-DAM and 6FDA/BPDA-DAM hollow fibers were successfully fabricated from a dry-jet/wet-quench spinning process. Polymer rigidity, glass-rubber transition and asymmetric morphology were correlated. CMS hollow fiber membranes produced from 6FDA-polymer precursors showed significant improvement in permeance for ethylene/ethane and propylene/propane separations. Further studies revealed that the CMS membranes are olefins-selective, which means the membranes are able to effectively separate olefins (ethylene and propylene) from paraffins (ethane and propane). This unique feature of CMS materials enables advanced hybrid membrane-distillation process designs. By using the olefins-selective membranes, these new processes may provide advantages over previously proposed retrofitting concepts. Further applications of the membranes are explored for hydrocarbons processes. Significant energy savings and even reduced footprint may be achieved in olefins production units. © 2012 Elsevier B.V.

Cell membrane temperature rate sensitivity predicted from the Nernst equation.

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Barnes, F S

1984-01-01

A hyperpolarized current is predicted from the Nernst equation for conditions of positive temperature derivatives with respect to time. This ion current, coupled with changes in membrane channel conductivities, is expected to contribute to a transient potential shift across the cell membrane for silent cells and to a change in firing rate for pacemaker cells.

Modal radiation patterns of baffled circular plates and membranes.

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Christiansen, Thomas Lehrmann; Hansen, Ole; Thomsen, Erik Vilain; Jensen, Jørgen Arendt

2014-05-01

The far field velocity potential and radiation pattern of baffled circular plates and membranes are found analytically using the full set of modal velocity profiles derived from the corresponding equation of motion. The derivation is valid for a plate or membrane subjected to an external excitation force, which is used as a sound receiver in any medium or as a sound transmitter in a gaseous medium. A general, concise expression is given for the radiation pattern of any mode of the membrane and the plate with arbitrary boundary conditions. Specific solutions are given for the four special cases of a plate with clamped, simply supported, and free edge boundary conditions as well as for the membrane. For all non-axisymmetric modes, the velocity potential along the axis of the radiator is found to be strictly zero. In the long wavelength limit, the radiation pattern of all axisymmetric modes approaches that of a monopole, while the non-axisymmetric modes exhibit multipole behavior. Numerical results are also given, demonstrating the implications of having non-axisymmetric excitation using both a point excitation with varying eccentricity and a homogeneous excitation acting on half of the circular radiator.

Membranolytic Activity of Bile Salts: Influence of Biological Membrane Properties and Composition

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Alfred Blume

2007-10-01

Full Text Available The two main steps of the membranolytic activity of detergents: 1 the partitioning of detergent molecules in the membrane and 2 the solubilisation of the membrane are systematically investigated. The interactions of two bile salt molecules, sodium cholate (NaC and sodium deoxycholate (NaDC with biological phospholipid model membranes are considered. The membranolytic activity is analysed as a function of the hydrophobicity of the bile salt, ionic strength, temperature, membrane phase properties, membrane surface charge and composition of the acyl chains of the lipids. The results are derived from calorimetric measurements (ITC, isothermal titration calorimetry. A thermodynamic model is described, taking into consideration electrostatic interactions, which is used for the calculation of the partition coefficient as well as to derive the complete thermodynamic parameters describing the interaction of detergents with biological membranes (change in enthalpy, change in free energy, change in entropy etc. The solubilisation properties are described in a so-called vesicle-to-micelle phase transition diagram. The obtained results are supplemented and confirmed by data obtained from other biophysical techniques (DSC differential scanning calorimetry, DLS dynamic light scattering, SANS small angle neutron scattering.

Incorporation of stromal cell-derived factor-1 alpha in PCL/gelatin electrospun membranes for guided bone regeneration

NARCIS (Netherlands)

Ji, W.; Yang, F.; Ma, J.L.; Bouma, M.J.; Boerman, O.C.; Chen, Z.; Beucken, J.J.J.P van den; Jansen, J.A.

2013-01-01

The goal of this work was to evaluate the effect of membrane functionalization with a chemotactic factor on cell recruitment and bone formation in order to develop a bioactive membrane for guided bone regeneration (GBR) applications. To this end. GBR membranes were prepared by electrospinning using

A FTIR study water in membrane of nitrocellulose prepared by phase inversion

International Nuclear Information System (INIS)

Benosmane, N.; Boutemeur, B.; Hamdi, M.

2004-01-01

Full text.Cellulose derivates were the first biopolymers used to produce synthesis membranes for technical applications, in this study the state of water in asymmetric membrane of nitrocellulose, prepared by the phase inversion process, was investigated using infrared spectroscopy (FTIR), after membrane preparation by the wet inversion process in acetone, the spectre FTIR of wet asymmetric membrane of nitrocellulose after immersion in water (after one week) is compared to the spectre of dried asymmetric membrane of nitrocellulose, the difference in spectre of dried and wet membrane indicate a weakly hydrogen-bonded to the polymer hydroxyl groups between water and hydroxyl groups in surface of membrane, the results demonstrate the amount of water species present in the surface of asymmetric membrane and heterogeneous of surface

Black hole mass formula in the membrane paradigm

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Lemos, José P. S.; Zaslavskii, Oleg B.

2018-03-01

The membrane paradigm approach adopts a timelike surface, stretched out off the null event horizon, to study several important black hole properties. We use this powerful tool to give a direct derivation of the black hole mass formula in the static and stationary cases without and with electric field. Since here the membrane is a self-gravitating material system, we go beyond the usual applicability on test particles and test fields of the paradigm.

Biofouling in capillary and spiral wound membranes facilitated by marine algal bloom

NARCIS (Netherlands)

Villacorte, L.O.; Ekowati, Y.; Calix-Ponce, H.N.; Kisielius, V.; Kleijn, J.M.; Vrouwenvelder, J.S.; Schippers, J.C.; Kennedy, M.D.

2017-01-01

Algal-derived organic matter (AOM), particularly transparent exopolymer particles, has been suspected to facilitate biofilm development in membrane systems (e.g., seawater reverse osmosis). This study demonstrates the possible role of AOM on biofouling in membrane systems affected by marine algal

Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

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Roberts Paul C

2009-04-01

Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

Mass transfer rate through liquid membranes: interfacial chemical reactions and diffusion as simultaneous permeability controlling factors

International Nuclear Information System (INIS)

Danesi, P.R.; Horwitz, E.P.; Vandegrift, G.F.; Chiarizia, R.

1981-01-01

Equations describing the permeability of a liquid membrane to metal cations have been derived taking into account aqueous diffusion, membrane diffusion, and interfacial chemical reactions as simultaneous permeability controlling factors. Diffusion and chemical reactions have been coupled by a simple model analogous to the one previously described by us to represent liquid-liquid extraction kinetics. The derived equations, which make use of experimentally determined interfacial reaction mechanisms, qualitatively fit unexplained literature data regarding Cu 2+ transfer through liquid membranes. Their use to predict and optimize membrane permeability in practical separation processes by setting the appropriate concentration of the membrane carrier [LIX 64 (General Mills), a commercial β-hydroxy-oxime] and the pH of the aqueous copper feed solution is briefly discussed. 4 figures

Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

Energy Technology Data Exchange (ETDEWEB)

Muratore, Massimo, E-mail: M.Muratore@ed.ac.uk [Institute of Integrated Micro and Nano System, School of Engineering, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Mitchell, Steve [Institute of Molecular Plant Science, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Waterfall, Martin [Institute of Immunology and Infection Research, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JT (United Kingdom)

2013-09-06

Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

International Nuclear Information System (INIS)

Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

2013-01-01

Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy

Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

Science.gov (United States)

Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

2016-11-01

Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

Physicochemical characterization of cellulose nanocrystal and nanoporous self-assembled CNC membrane derived from Ceiba pentandra.

Science.gov (United States)

Mohamed, Mohamad Azuwa; W Salleh, W N; Jaafar, Juhana; Ismail, A F; Abd Mutalib, Muhazri; Mohamad, Abu Bakar; M Zain, M F; Awang, Nor Asikin; Mohd Hir, Zul Adlan

2017-02-10

This research involves the rare utilisation of the kapok fibre (Ceiba pentandra) as a raw material for the fabrication of cellulose nanocrystal (CNC) and self-assembled CNC membranes. The isolation of CNC from Ceiba pentandra began with the extraction of cellulose via the chemical alkali extraction by using 5wt% NaOH, followed by the typical acidified bleaching method and, finally, the CNC production through acid hydrolysis with 60wt% H 2 SO 4 at the optimum time of 60min. The prepared CNC was then employed for the preparation of self-assembled membrane through the water suspension casting evaporation technique. The obtained CNC membrane was characterised in terms of its composition, crystallinity, thermal stability, as well as, structural and morphological features with the use of several techniques including FTIR, XRD, AFM, TEM, FESEM, and TGA. The FESEM and AFM analyses had illustrated the achievement of a self-assembled CNC membrane with a smooth surface and a well-distributed nano-porous structure, with the porosity of 52.82±7.79%. In addition, the findings proved that the self-assembled CNC membrane displayed good adsorption capability indicated by the recorded efficiency of 79% and 85% for 10mg/L and 5mg/L of methylene blue in an aqueous solution, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

Science.gov (United States)

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane.

Science.gov (United States)

Witschas, Katja; Jobin, Marie-Lise; Korkut, Dursun Nizam; Vladan, Maria Magdalena; Salgado, Gilmar; Lecomte, Sophie; Vlachova, Viktorie; Alves, Isabel D

2015-05-01

The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific. Copyright © 2015 Elsevier B.V. All rights reserved.

N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

Science.gov (United States)

Shaikh, Saame Raza; Teague, Heather

2012-12-01

This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Unraveling the role of membrane microdomains during microbial infections.

Science.gov (United States)

Bagam, Prathyusha; Singh, Dhirendra P; Inda, Maria Eugenia; Batra, Sanjay

2017-10-01

Infectious diseases pose major socioeconomic and health-related threats to millions of people across the globe. Strategies to combat infectious diseases derive from our understanding of the complex interactions between the host and specific bacterial, viral, and fungal pathogens. Lipid rafts are membrane microdomains that play important role in life cycle of microbes. Interaction of microbial pathogens with host membrane rafts influences not only their initial colonization but also their spread and the induction of inflammation. Therefore, intervention strategies aimed at modulating the assembly of membrane rafts and/or regulating raft-directed signaling pathways are attractive approaches for the. management of infectious diseases. The current review discusses the latest advances in terms of techniques used to study the role of membrane microdomains in various pathological conditions and provides updated information regarding the role of membrane rafts during bacterial, viral and fungal infections.

Radiation-grafted membranes based on polyethylene for direct methanol fuel cells

Energy Technology Data Exchange (ETDEWEB)

Sherazi, Tauqir A. [Department of Chemistry, Government College University, Lahore 54000 (Pakistan); Institute for Chemical Process and Environmental Technology, National Research Council Canada, 1200 Montreal Road, Ottawa, ON K1A 0R6 (Canada); Guiver, Michael D.; Kingston, David; Xue, Xinzhong [Institute for Chemical Process and Environmental Technology, National Research Council Canada, 1200 Montreal Road, Ottawa, ON K1A 0R6 (Canada); Ahmad, Shujaat [PIEAS/PINSTECH, P O Nilore, Islamabad 45650 (Pakistan); Kashmiri, M. Akram [Department of Chemistry, Government College University, Lahore 54000 (Pakistan); Board of Intermediate and Secondary Education, Lahore 54000 (Pakistan)

2010-01-01

Styrene was grafted onto ultrahigh molecular weight polyethylene powder (UHMWPE) by gamma irradiation using a {sup 60}Co source. Compression moulded films of selected pre-irradiated styrene-grafted ultrahigh molecular weight polyethylene (UHMWPE-g-PS) were post-sulfonated to the sulfonic acid derivative (UHMWPE-g-PSSA) for use as proton exchange membranes (PEMs). The sulfonation was confirmed by X-ray photoelectron spectroscopy (XPS). The melting and flow properties of UHMWPE and UHMWPE-g-PS are conducive to forming homogeneous pore-free membranes. Both the ion conductivity and methanol permeability coefficient increased with degree of grafting, but the grafted membranes showed comparable or higher ion conductivity and lower methanol permeability than Nafion {sup registered} 117 membrane. One UHMWPE-g-PS membrane was fabricated into a membrane-electrode assembly (MEA) and tested as a single cell direct methanol fuel cell (DMFC). Low membrane cost and acceptable fuel cell performance indicate that UHMWPE-g-PSSA membranes could offer an alternative approach to perfluorosulfonic acid-type membranes for DMFC. (author)

Computer simulations of phospholipid - membrane thermodynamic fluctuations

DEFF Research Database (Denmark)

Pedersen, U.R.; Peters, Günther H.j.; Schröder, T.B.

2008-01-01

This paper reports all-atom computer simulations of five phospholipid membranes, DMPC, DPPC, DMPG, DMPS, and DMPSH, with a focus on the thermal equilibrium fluctuations of volume, energy, area, thickness, and order parameter. For the slow fluctuations at constant temperature and pressure (defined...... membranes, showing a similar picture. The cause of the observed strong correlations is identified by splitting volume and energy into contributions from tails, heads, and water, showing that the slow volume-energy fluctuations derive from the tail region’s van der Waals interactions and are thus analogous...

Crosslinked cellulose thin film composite nanofiltration membranes with zero salt rejection

KAUST Repository

Puspasari, Tiara

2015-05-14

We report a new synthetic route of fabricating regenerated cellulose nanofiltration membranes. The membranes are composite membranes with a thin selective layer of cellulose, which was prepared by regeneration of trimethylsilyl cellulose (a hydrophobic cellulose derivative) film followed by crosslinking. Filtration experiments using mixtures of sugar and sodium chloride showed that solutes above 300 Da were highly rejected whereas practically no rejection was observed for NaCl. This is a big advantage for a complete desalination as the existing commercial nanofiltration membranes typically exhibit NaCl rejection in the range of 30–60%. Membranes with zero NaCl rejection are required for recovery and purification applications in food, chemical and pharmaceutical industry.

Cell membrane softening in human breast and cervical cancer cells

Science.gov (United States)

Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

2015-08-01

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

Highly efficient transition metal and nitrogen co-doped carbide-derived carbon electrocatalysts for anion exchange membrane fuel cells

Science.gov (United States)

Ratso, Sander; Kruusenberg, Ivar; Käärik, Maike; Kook, Mati; Puust, Laurits; Saar, Rando; Leis, Jaan; Tammeveski, Kaido

2018-01-01

The search for an efficient electrocatalyst for oxygen reduction reaction (ORR) to replace platinum in fuel cell cathode materials is one of the hottest topics in electrocatalysis. Among the many non-noble metal catalysts, metal/nitrogen/carbon composites made by pyrolysis of cheap materials are the most promising with control over the porosity and final structure of the catalyst a crucial point. In this work we show a method of producing a highly active ORR catalyst in alkaline media with a controllable porous structure using titanium carbide derived carbon as a base structure and dicyandiamide along with FeCl3 or CoCl2 as the dopants. The resulting transition metal-nitrogen co-doped carbide derived carbon (M/N/CDC) catalyst is highly efficient for ORR electrocatalysis with the activity in 0.1 M KOH approaching that of commercial 46.1 wt.% Pt/C. The catalyst materials are also investigated by scanning electron microscopy, Raman spectroscopy and X-ray photoelectron spectroscopy to characterise the changes in morphology and composition causing the raise in electrochemical activity. MEA performance of M/N/CDC cathode materials in H2/O2 alkaline membrane fuel cell is tested with the highest power density reached being 80 mW cm-2 compared to 90 mW cm-2 for Pt/C.

Technical Performance and Economic Evaluation of Evaporative and Membrane-Based Concentration for Biomass-Derived Sugars

International Nuclear Information System (INIS)

Sievers, David A.; Stickel, Jonathan J.; Grundl, Nicholas J.; Tao, Ling

2017-01-01

Several conversion pathways of lignocellulosic biomass to advanced biofuels require or benefit from using concentrated sugar syrups of 600 g/L or greater. And while concentration may seem straightforward, thermal sugar degradation and energy efficiency remain major concerns. This study evaluated the trade-offs in product recovery, energy consumption, and economics between evaporative and membrane-based concentration methods. The degradation kinetics of xylose and glucose were characterized and applied to an evaporator process simulation. Though significant sugar loss was predicted for certain scenarios due to the Maillard reaction, industrially common falling-film plate evaporators offer short residence times (<5 min) and are expected to limit sugar losses. Membrane concentration experiments characterized flux and sugar rejection, but diminished flux occurred at >100 g/L. A second step using evaporation is necessary to achieve target concentrations. Techno-economic process model simulations evaluated the overall economics of concentrating a 35 g/L sugar stream to 600 g/L in a full-scale biorefinery. A two-step approach of preconcentrating using membranes and finishing with an evaporator consumed less energy than evaporation alone but was more expensive because of high capital expenses of the membrane units.

Assay of flippase activity in proteoliposomes using fluorescent lipid derivatives

DEFF Research Database (Denmark)

Marek, Magdalena; Günther-Pomorski, Thomas

2016-01-01

Specific membrane proteins, termed lipid flippases, play a central role in facilitating the movement of lipids across cellular membranes. In this protocol, we describe the reconstitution of ATP-driven lipid flippases in liposomes and the analysis of their in vitro flippase activity based on the use...... of fluorescent lipid derivatives. Working with purified and reconstituted systems provides a well-defined experimental setup and allows to directly characterize these membrane proteins at the molecular level....

Planar, Polysilazane-Derived Porous Ceramic Supports for Membrane and Catalysis Applications.

Science.gov (United States)

Konegger, Thomas; Williams, Lee F; Bordia, Rajendra K

2015-10-01

Porous, silicon carbonitride-based ceramic support structures for potential membrane and catalysis applications were generated from a preceramic polysilazane precursor in combination with spherical, ultrahigh-molecular weight polyethylene microparticles through a sacrificial filler approach. A screening evaluation was used for the determination of the impact of both porogen content and porogen size on pore structure, strength, and permeability characteristics of planar specimens. By optimizing both the composition as well as cross-linking parameters, maximum characteristic biaxial flexural strengths of 65 MPa and porosities of 42% were achieved. The evolution of an interconnected, open-pore network during thermal porogen removal and conversion of the preceramic polymer led to air permeabilities in the order of 10 -14 m 2 . The materials were further exposed to long-term heat treatments to demonstrate the stability of properties after 100 h at 800°C in oxidizing, inert, and reducing environments. The determined performance, in combination with the versatile preparation method, illustrates the feasibility of this processing approach for the generation of porous ceramic support structures for applications at elevated temperatures in a variety of fields, including membrane and catalysis science.

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets.

Science.gov (United States)

Ryu, Seungmi; Yoo, Jin; Jang, Yeongseon; Han, Jin; Yu, Seung Jung; Park, Jooyeon; Jung, Seon Yeop; Ahn, Kyung Hyun; Im, Sung Gap; Char, Kookheon; Kim, Byung-Soo

2015-10-27

Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells.

High pressure pure- and mixed-gas separation of CO2/CH4 by thermally-rearranged and carbon molecular sieve membranes derived from a polyimide of intrinsic microporosity

KAUST Repository

Swaidan, Raja

2013-11-01

Natural gas sweetening, one of the most promising venues for the growth of the membrane gas separation industry, is dominated by polymeric materials with relatively low permeabilities and moderate selectivities. One strategy towards improving the gas transport properties of a polymer is enhancement of microporosity either by design of polymers of intrinsic microporosity (PIMs) or by thermal treatment of polymeric precursors. For the first time, the mixed-gas CO2/CH4 transport properties are investigated for a complete series of thermally-rearranged (TR) (440°C) and carbon molecular sieve (CMS) membranes (600, 630 and 800°C) derived from a polyimide of intrinsic microporosity (PIM-6FDA-OH). The pressure dependence of permeability and selectivity is reported up to 30bar for 1:1, CO2:CH4 mixed-gas feeds at 35°C. The TR membrane exhibited ~15% higher CO2/CH4 selectivity relative to pure-gas feeds due to reductions in mixed-gas CH4 permeability reaching 27% at 30bar. This is attributed to increased hindrance of CH4 transport by co-permeation of CO2. Interestingly, unusual increases in mixed-gas CH4 permeabilities relative to pure-gas values were observed for the CMS membranes, resulting in up to 50% losses in mixed-gas selectivity over the applied pressure range. © 2013 Elsevier B.V.

Facilitated transport of HNO3 through a supported liquid membrane containing a tertiary amine as carrier

International Nuclear Information System (INIS)

Cianetti, C.; Danesi, P.R.

1983-01-01

The facilitated transport of HNO 3 through a supported liquid membrane consisting of a porous polypropylene film containing a solution of trilaurylamine in diethylbenzene as carrier was studied as a function of the stirring speed of the aqueous solutions and the membrane composition. A physico-chemical model which takes into account diffusion through an aqueous boundary layer, a fast interfacial chemical reaction leading to the formation of a membrane soluble alkylammonium salt and diffusion through the membrane was proposed. In this way, equations were derived which describe how composition changes, occurring in the course of the permeation process, influence the membrane permeability. The experimental data were quantitatively explained by the derived equations. The results indicate that the monomeric form of the trilaurylammonium nitrate salt is the species which is mainly responsible for the acid transport through the membrane. The diffusion coefficient of the permeating species and the order of magnitude of the thickness of the aqueous boundary layer were evaluated. 8 figures

Planar, Polysilazane?Derived Porous Ceramic Supports for Membrane and Catalysis Applications

OpenAIRE

Konegger, Thomas; Williams, Lee F.; Bordia, Rajendra K.

2015-01-01

Porous, silicon carbonitride?based ceramic support structures for potential membrane and catalysis applications were generated from a preceramic polysilazane precursor in combination with spherical, ultrahigh?molecular weight polyethylene microparticles through a sacrificial filler approach. A screening evaluation was used for the determination of the impact of both porogen content and porogen size on pore structure, strength, and permeability characteristics of planar specimens. By optimizin...

Mesenchymal Stem/Progenitor Cells Derived from Articular Cartilage, Synovial Membrane and Synovial Fluid for Cartilage Regeneration: Current Status and Future Perspectives.

Science.gov (United States)

Huang, Yi-Zhou; Xie, Hui-Qi; Silini, Antonietta; Parolini, Ornella; Zhang, Yi; Deng, Li; Huang, Yong-Can

2017-10-01

Large articular cartilage defects remain an immense challenge in the field of regenerative medicine because of their poor intrinsic repair capacity. Currently, the available medical interventions can relieve clinical symptoms to some extent, but fail to repair the cartilaginous injuries with authentic hyaline cartilage. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stem/progenitor cells with or without scaffolds. Mesenchymal stem/progenitor cells are promising graft cells for tissue regeneration, but the most suitable source of cells for cartilage repair remains controversial. The tissue origin of mesenchymal stem/progenitor cells notably influences the biological properties and therapeutic potential. It is well known that mesenchymal stem/progenitor cells derived from synovial joint tissues exhibit superior chondrogenic ability compared with those derived from non-joint tissues; thus, these cell populations are considered ideal sources for cartilage regeneration. In addition to the progress in research and promising preclinical results, many important research questions must be answered before widespread success in cartilage regeneration is achieved. This review outlines the biology of stem/progenitor cells derived from the articular cartilage, the synovial membrane, and the synovial fluid, including their tissue distribution, function and biological characteristics. Furthermore, preclinical and clinical trials focusing on their applications for cartilage regeneration are summarized, and future research perspectives are discussed.

Arct'Alg release from hydrogel membranes

International Nuclear Information System (INIS)

Amaral, Renata H.; Rogero, Sizue O.; Shihomatsu, Helena M.; Lugao, Ademar B.

2009-01-01

The hydrogel properties make them attractive for a variety of biomedical and pharmaceutical applications, primarily in drug delivery system. Synthetic hydrogels have been studied to develop new devices for drugs or cosmetic active agents release. Arct'Alg R is an extract derived from red algae biomass which has antioxidant, anti-inflammatory and tissue regeneration stimulant properties. This extract was incorporated to poly(N-vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA) hydrogel membranes obtained by gamma rays crosslinking technique. The ionizing radiation presents the advantage to occur polymerization and sterilization simultaneously in the same process. The aim of this work was the in vitro release kinetic study of Arct'Alg R from hydrogel membranes during 24 hours to verify the possibility of use in cosmetic and dermatological treatments. Results showed that about 50% and 30% of incorporated Arct'Alg R was released from PVP and PVA hydrogel membrane devices respectively. (author)

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

Science.gov (United States)

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Mimicking the effect of gravity using an elastic membrane

International Nuclear Information System (INIS)

Wu, Yecun; Zhu, Changqing; Wang, Yijun; Shi, Qingfan

2014-01-01

Comparing astrospace with an elastic membrane is an interesting analogy but it lacks a theoretical basis and experimental support. We develop a theoretical model that brings to light the relationship between the conceptual model of a gravity well and an elastic deformation equation of a membrane supporting a heavy ball, and further derive the ‘gravitational constant’ for such a small ‘elastic space’. The experimental data obtained are consistent with the prediction of our model, in mimicking the revolution of a small planet. Teaching practice shows that using an elastic membrane is a simple, intuitive and reliable method to enhance the quality of learning about the effect of gravity. (paper)

Cross flow microfiltration of oil-water emulsions using clay based ceramic membrane support and TiO2 composite membrane

Directory of Open Access Journals (Sweden)

Kanchapogu Suresh

2017-09-01

Full Text Available The main objective of this work is to study the effect of cross flow filtration conditions on the separation of oily wastewater using ceramic support and TiO2 membrane. Firstly, the low cost clay based ceramic membrane support was prepared by uniaxial compaction method using combination of pyrophyllite, quartz, feldspar, kaolin, ball clay and calcium carbonate along with PVA as a binder. Subsequently, TiO2 composite membrane was fabricated via hydrothermal route employing TiO2 sol derived from TiCl4 and NH4OH solution. Cross flow microfiltration investigations were carried out by utilizing oil-water emulsion concentration of 200 mg/L at three distinct applied pressures (69–207 kPa and three cross flow velocities (0.0885, 0.1327, and 0.1769 m/s. Compared to ceramic support, TiO2 composite membrane demonstrates better performance in terms of flux and removal efficiency of oil and also the rate of flux decline during filtration operation is lower due to highly hydrophilic surface of the TiO2 membrane. TiO2 membrane displays the oil removal efficiency of 99% in the entire range of applied pressures investigation, while ceramic support shows 93–96% of oil removal.

Modeling electrically active viscoelastic membranes.

Directory of Open Access Journals (Sweden)

Sitikantha Roy

Full Text Available The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism.

Thermally rearranged (TR) bismaleimide-based network polymers for gas separation membranes.

Science.gov (United States)

Do, Yu Seong; Lee, Won Hee; Seong, Jong Geun; Kim, Ju Sung; Wang, Ho Hyun; Doherty, Cara M; Hill, Anita J; Lee, Young Moo

2016-11-15

Highly permeable, thermally rearranged polymer membranes based on bismaleimide derivatives that exhibit excellent CO 2 permeability up to 5440 Barrer with a high BET surface area (1130 m 2 g -1 ) are reported for the first time. In addition, the membranes can be easily used to form semi-interpenetrating networks with other polymers endowing them with superior gas transport properties.

Carbon molecular sieve membranes derived from Matrimid® polyimide for nitrogen/methane separation

KAUST Repository

Ning, Xue

2014-01-01

A commercial polyimide, Matrimid® 5218, was pyrolyzed under an inert argon atmosphere to produce carbon molecular sieve (CMS) dense film membranes for nitrogen/methane separation. The resulting CMS dense film separation performance was evaluated using both pure and mixed N2/CH4 permeation tests. The effects of final pyrolysis temperature on N 2/CH4 separation are reported. The separation performance of all CMS dense films significantly exceeds the polymer precursor dense film. The CMS dense film pyrolyzed at 800 C shows very attractive separation performance that surpasses the polymer membrane upper bound line, with N 2 permeability of 6.8 Barrers and N2/CH4 permselectivity of 7.7 from pure gas permeation, and N2 permeability of 5.2 Barrers and N2/CH4 permselectivity of 6.0 from mixed gas permeation. The temperature dependences of permeabilities, sorption coefficients, and diffusion coefficients of the membrane were studied, and the activation energy for permeation and diffusion, as well as the apparent heats of sorption are reported. The high permselectivity of this dense film is shown to arise from a significant entropic contribution in the diffusion selectivity. The study shows that the rigid \\'slit-shaped\\' CMS pore structure can enable a strong molecular sieving effect to effectively distinguish the size and shape difference between N2 and CH4. © 2013 Elsevier Ltd. All rights reserved.

Development of a pH sensing membrane electrode based on a new calix[4]arene derivative.

Science.gov (United States)

Kormalı Ertürün, H Elif; Demirel Özel, Ayça; Sayın, Serkan; Yılmaz, Mustafa; Kılıç, Esma

2015-01-01

A new pH sensing poly(vinyl chloride) (PVC) membrane electrode was developed by using recently synthesized 5,17-bis(4-benzylpiperidine-1-yl)methyl-25,26,27,28-tetrahydroxy calix[4]arene as an ionophore. The effects of membrane composition, inner filling solution and conditioning solution on the potential response of the proposed pH sensing membrane electrode were investigated. An optimum membrane composition of 3% ionophore, 67% o-nitrophenyl octyl ether (o-NPOE) as plasticizer, 30% PVC was found. The electrode exhibited a near-Nernstian slope of 58.7±1.1 mV pH(-1) in the pH range 1.9-12.7 at 20±1 °C. It showed good selectivity for H(+) ions in the presence of some cations and anions and a longer lifetime of at least 12 months when compared with the other PVC membrane pH electrodes reported in the literature. Having a wide working pH range, it was not only applied as a potentiometric indicator electrode in various acid-base titrations, but also successfully employed in different real samples. It has good reproducibility and repeatability with a response time of 6-7s. Compared to traditional glass pH electrode, it exhibited excellent potentiometric response after being used in fluoride-containing media. Copyright © 2014 Elsevier B.V. All rights reserved.

Biofouling in capillary and spiral wound membranes facilitated by marine algal bloom

DEFF Research Database (Denmark)

Villacorte, L.O.; Ekowati, Y.; Calix-Ponce, H.N.

2017-01-01

blooms. The tendency of AOM from bloom-forming marine algae to adhere to membranes and its ability to enhance biofilm growth were measured using atomic force microscopy, flow cytometry, liquid chromatography and accelerated membrane biofouling experiments. Adhesion force measurements indicate that AOM......Algal-derived organic matter (AOM), particularly transparent exopolymer particles, has been suspected to facilitate biofilm development in membrane systems (e.g., seawater reverse osmosis). This study demonstrates the possible role of AOM on biofouling in membrane systems affected by marine algal...... biodegradable nutrients. The abovementioned findings indicate that AOM facilitates the onset of membrane biofouling primarily as a conditioning platform and to some extent as a nutrient source for biofilm-forming bacteria....

Sulfonated graphene oxide/nafion composite membrane for vanadium redox flow battery.

Science.gov (United States)

Kim, Byung Guk; Han, Tae Hee; Cho, Chang Gi

2014-12-01

Nafion is the most frequently used as the membrane material due to its good proton conductivity, and excellent chemical and mechanical stabilities. But it is known to have poor barrier property due to its well-developed water channels. In order to overcome this drawback, graphene oxide (GO) derivatives were introduced for Nafion composite membranes. Sulfonated graphene oxide (sGO) was prepared from GO. Both sGO and GO were treated each with phenyl isocyanate and transformed into corresponding isGO and iGO in order to promote miscibility with Nafion. Then composite membranes were obtained, and the adaptability as a membrane for vanadium redox flow battery (VRFB) was investigated in terms of proton conductivity and vanadium permeability. Compared to a pristine Nafion, proton conductivities of both isGO/Nafion and iGO/Nafion membranes showed less temperature sensitivity. Both membranes also showed quite lower vanadium permeability at room temperature. Selectivity of the membrane was the highest for isGO/Nafion and the lowest for the pristine Nafion.

A theoretical model for gas permeability in a composite membrane

International Nuclear Information System (INIS)

Serrano, D. A

2009-01-01

We present in this work an analytical expression for permeability in a two-layer composite membrane, which was derived assuming the same hypothesis as those of Adzumi model for permeability in a homogeneous membrane. Whereas in Adzumi model permeability shows a linear dependence on the mean pressure, our model for a composite membrane related permeability to pressure through a rather complex expression, which covers the whole range of flow, from molecular-Knudsen to viscous-Poiseuille regimes. The expression obtained for permeability contained information of membrane structural properties as pore size, porosity and thickness of each layer, as well as gas nature and operational conditions. Our two-layer-model expression turns into Adzumi formula when the structure of the layers approach to each other. [es

Active fragments from pro- and antiapoptotic BCL-2 proteins have distinct membrane behavior reflecting their functional divergence.

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Yannis Guillemin

Full Text Available BACKGROUND: The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. CONCLUSION/SIGNIFICANCE: BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains.

ZIF-78 membrane derived from amorphous precursors with permselectivity for cyclohexanone/cyclohexanol mixture

KAUST Repository

Fan, Lili

2014-07-01

Cyclohexanone and cyclohexanol are products of selective oxidation of cyclohexane. They are important industrial intermediates and difficult to be separated due to their close boiling points. In this work, well-intergrown ZIF-78 membrane was successfully synthesized on the porous silica substrate by secondary growth method and applied for separation of cyclohexanone/cyclohexanol mixture for the first time. Meanwhile, a facile method for seeding procedure was developed by utilizing the amorphous ZIF-78 precursors to provide better-distributed nucleation sites. Both XRD and SEM results confirmed the good quality of the membrane. The pervaporation separation of cyclohexanone/ cyclohexanol mixture were carried out at room temperature with permselectivity of 1:2 and total flux around 8.7 × 10-2 kg m-2 h-1. © 2013 Elsevier Inc. All rights reserved.

Fluorescent probes for detecting cholesterol-rich ordered membrane microdomains: entangled relationships between structural analogies in the membrane and functional homologies in the cell

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Gérald Gaibelet

2017-02-01

Full Text Available This review addresses the question of fluorescent detection of ordered membrane (micro domains in living (cultured cells, with a “practical” point of view since the situation is much more complicated than for studying model membranes. We first briefly recall the bases of model membrane structural organization involving liquid-ordered and -disordered phases, and the main features of their counterparts in cell membranes that are the various microdomains. We then emphasize the utility of the fluorescent probes derived from cholesterol, and delineate the respective advantages, limitations and drawbacks of the existing ones. In particular, besides their intra-membrane behavior, their relevant characteristics should integrate their different cellular fates for membrane turn-over, trafficking and metabolism, in order to evaluate and improve their efficiency for in-situ probing membrane microdomains in the cell physiology context. Finally, at the present stage, it appears that Bdp-Chol and Pyr-met-Chol display well complementary properties, allowing to use them in combination to improve the reliability of the current experimental approaches. But the field is still open, and there remains much work to perform in this research area.

Carbon molecular sieve membranes derived from Matrimid® polyimide for nitrogen/methane separation

KAUST Repository

Ning, Xue; Koros, William J.

2014-01-01

A commercial polyimide, Matrimid® 5218, was pyrolyzed under an inert argon atmosphere to produce carbon molecular sieve (CMS) dense film membranes for nitrogen/methane separation. The resulting CMS dense film separation performance was evaluated

Hamilton's equations for a fluid membrane

International Nuclear Information System (INIS)

Capovilla, R; Guven, J; Rojas, E

2005-01-01

Consider a homogeneous fluid membrane described by the Helfrich-Canham energy, quadratic in the mean curvature of the membrane surface. The shape equation that determines equilibrium configurations is fourth order in derivatives and cubic in the mean curvature. We introduce a Hamiltonian formulation of this equation which dismantles it into a set of coupled first-order equations. This involves interpreting the Helfrich-Canham energy as an action; equilibrium surfaces are generated by the evolution of space curves. Two features complicate the implementation of a Hamiltonian framework. (i) The action involves second derivatives. This requires treating the velocity as a phase-space variable and the introduction of its conjugate momentum. The canonical Hamiltonian is constructed on this phase space. (ii) The action possesses a local symmetry-reparametrization invariance. The two labels we use to parametrize points on the surface are themselves physically irrelevant. This symmetry implies primary constraints, one for each label, that need to be implemented within the Hamiltonian. The two Lagrange multipliers associated with these constraints are identified as the components of the acceleration tangential to the surface. The conservation of the primary constraints implies two secondary constraints, fixing the tangential components of the momentum conjugate to the position. Hamilton's equations are derived and the appropriate initial conditions on the phase-space variables are identified. Finally, it is shown how the shape equation can be reconstructed from these equations

Self-assembling peptides form nanodiscs that stabilize membrane proteins

DEFF Research Database (Denmark)

Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

2014-01-01

-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

Stringy and Membranic Theory of Swimming of Micro-organisms

OpenAIRE

Kawamura, Masako; Nojiri, Shin'ichi; Sugamoto, Akio

1996-01-01

When the swimming of micro-organisms is viewed from the string and membrane theories coupled to the velocity field of the fluid, a number of interesting results are derived; 1) importance of the area (or volume) preserving algebra, 2) usefulness of the $N$-point Reggeon (membranic) amplitudes, and of the gas to liquid transition in case of the red tide issues, 3) close relation between the red tide issue and the generation of Einstein gravity, and 4) possible understanding of the three differ...

Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

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Dasgupta, Sabyasachi; Auth, Thorsten; Gov, Nir S.; Satchwell, Timothy J.; Hanssen, Eric; Zuccala, Elizabeth S.; Riglar, David T.; Toye, Ashley M.; Betz, Timo; Baum, Jake; Gompper, Gerhard

2014-01-01

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells. PMID:24988340

A comparison of micropore membrane inlet mass spectrometry-derived pulmonary shunt measurement with Riley shunt in a porcine model.

Science.gov (United States)

Duenges, Bastian; Vogt, Andreas; Bodenstein, Marc; Wang, Hemei; Böhme, Stefan; Röhrig, Bernd; Baumgardner, James E; Markstaller, Klaus

2009-12-01

The multiple inert gas elimination technique was developed to measure shunt and the ratio of alveolar ventilation to simultaneous alveolar capillary blood flow in any part of the lung (V(A)'/Q') distributions. Micropore membrane inlet mass spectrometry (MMIMS), instead of gas chromatography, has been introduced for inert gas measurement and shunt determination in a rabbit lung model. However, agreement with a frequently used and accepted method for quantifying deficits in arterial oxygenation has not been established. We compared MMIMS-derived shunt (M-S) as a fraction of total cardiac output (CO) with Riley shunt (R-S) derived from the R-S formula in a porcine lung injury model. To allow a broad variance of atelectasis and therefore shunt fraction, 8 sham animals did not receive lavage, and 8 animals were treated by lung lavages with 30 mL/kg warmed lactated Ringer's solution as follows: 2 animals were lavaged once, 5 animals twice, and 1 animal 3 times. Variables were recorded at baseline and twice after induction of lung injury (T1 and T2). Retention data of sulfur hexafluoride, krypton, desflurane, enflurane, diethyl ether, and acetone were analyzed by MMIMS, and M-S was derived using a known algorithm for the multiple inert gas elimination technique. Standard formulas were used for the calculation of R-S. Forty-four pairs of M-S and R-S were recorded. M-S ranged from 0.1% to 35.4% and R-S from 3.7% to 62.1%. M-S showed a correlation with R-S described by linear regression: M-S = -4.26 + 0.59 x R-S (r(2) = 0.83). M-S was on average lower than R-S (mean = -15.0% CO, sd = 6.5% CO, and median = -15.1), with lower and upper limits of agreement of -28.0% and -2.0%, respectively. The lower and upper limits of the 95% confidence intervals were -17.0 and -13.1 (P < 0.001, Student's t-test). Shunt derived from MMIMS inert gas retention data correlated well with R-S during breathing of oxygen. Shunt as derived by MMIMS was generally less than R-S.

The proteome of neurofilament-containing protein aggregates in blood

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Rocco Adiutori

2018-07-01

Full Text Available Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf, the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP, for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS. Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

Modelling the host-pathogen interactions of macrophages and Candida albicans using Game Theory and dynamic optimization.

Science.gov (United States)

Dühring, Sybille; Ewald, Jan; Germerodt, Sebastian; Kaleta, Christoph; Dandekar, Thomas; Schuster, Stefan

2017-07-01

The release of fungal cells following macrophage phagocytosis, called non-lytic expulsion, is reported for several fungal pathogens. On one hand, non-lytic expulsion may benefit the fungus in escaping the microbicidal environment of the phagosome. On the other hand, the macrophage could profit in terms of avoiding its own lysis and being able to undergo proliferation. To analyse the causes of non-lytic expulsion and the relevance of macrophage proliferation in the macrophage- Candida albicans interaction, we employ Evolutionary Game Theory and dynamic optimization in a sequential manner. We establish a game-theoretical model describing the different strategies of the two players after phagocytosis. Depending on the parameter values, we find four different Nash equilibria and determine the influence of the systems state of the host upon the game. As our Nash equilibria are a direct consequence of the model parameterization, we can depict several biological scenarios. A parameter region, where the host response is robust against the fungal infection, is determined. We further apply dynamic optimization to analyse whether macrophage mitosis is relevant in the host-pathogen interaction of macrophages and C. albicans For this, we study the population dynamics of the macrophage- C. albicans interactions and the corresponding optimal controls for the macrophages, indicating the best macrophage strategy of switching from proliferation to attacking fungal cells. © 2017 The Author(s).

Evidence of the presence of a functional Dot/Icm type IV-B secretion system in the fish bacterial pathogen Piscirickettsia salmonis.

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Fernando A Gómez

Full Text Available Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.

Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms.

Science.gov (United States)

Yamanaka, Takeshi; Yamane, Kazuyoshi; Furukawa, Tomoyo; Matsumoto-Mashimo, Chiho; Sugimori, Chieko; Nambu, Takayuki; Obata, Noboru; Walker, Clay B; Leung, Kai-Poon; Fukushima, Hisanori

2011-08-25

Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built. This study evaluates the role of EPS in Prevotella intermedia for the expression of virulence. This evaluation was accomplished by comparing EPS-producing P. intermedia strains 17 and OD1-16 with non-producing P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis. EPS-producing P. intermedia strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 107 colony-forming units (CFU). In comparison, P. intermedia ATCC 25611 and P. gingivalis ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (109 CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, P. intermedia strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes. These results demonstrate that the production of EPS by P. intermedia strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.

The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress.

Science.gov (United States)

Mancini, Stefano; Imlay, James A

2015-05-01

Hydrogen peroxide pervades many natural environments, including the phagosomes that mediate cell-based immunity. Transcriptomic analysis showed that during protracted low-grade H(2)O(2) stress, Escherichia coli responds by activating both the OxyR defensive regulon and the Fur iron-starvation response. OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). Mutations that blocked either adaptation caused the accumulation of porphyrin intermediates, inadequate activation of heme enzymes, low catalase activity, defective clearance of H(2)O(2) and a failure to grow. Genetic analysis indicated that HemH induction is needed to compensate for iron sequestration by the mini-ferritin Dps. Dps activity protects DNA and proteins by limiting Fenton chemistry, but it interferes with the ability of HemH to acquire the iron that it needs to complete heme synthesis. HemF is a manganoprotein that displaces HemN, an iron-sulfur enzyme whose synthesis and/or stability is apparently problematic during H(2)O(2) stress. Thus, the primary responses to H(2)O(2), including the sequestration of iron, require compensatory adjustments in the mechanisms of iron-cofactor synthesis. The results support the growing evidence that oxidative stress is primarily an iron pathology. © 2015 John Wiley & Sons Ltd.

Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

Science.gov (United States)

Verma, Jitender Kumar; Rastogi, Ruchir

2017-01-01

Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Erik T Sakowski

2015-07-01

Full Text Available The success of Mycobacterium tuberculosis (Mtb as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1 promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.

Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

Science.gov (United States)

Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

2015-02-01

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. © FASEB.

Pathology and immune reactivity: understanding multidimensionality in pulmonary tuberculosis.

Science.gov (United States)

Dorhoi, Anca; Kaufmann, Stefan H E

2016-03-01

Heightened morbidity and mortality in pulmonary tuberculosis (TB) are consequences of complex disease processes triggered by the causative agent, Mycobacterium tuberculosis (Mtb). Mtb modulates inflammation at distinct stages of its intracellular life. Recognition and phagocytosis, replication in phagosomes and cytosol escape induce tightly regulated release of cytokines [including interleukin (IL)-1, tumor necrosis factor (TNF), IL-10], chemokines, lipid mediators, and type I interferons (IFN-I). Mtb occupies various lung lesions at sites of pathology. Bacteria are barely detectable at foci of lipid pneumonia or in perivascular/bronchiolar cuffs. However, abundant organisms are evident in caseating granulomas and at the cavity wall. Such lesions follow polar trajectories towards fibrosis, encapsulation and mineralization or liquefaction, extensive matrix destruction, and tissue injury. The outcome is determined by immune factors acting in concert. Gradients of cytokines and chemokines (CCR2, CXCR2, CXCR3/CXCR5 agonists; TNF/IL-10, IL-1/IFN-I), expression of activation/death markers on immune cells (TNF receptor 1, PD-1, IL-27 receptor) or abundance of enzymes [arginase-1, matrix metalloprotease (MMP)-1, MMP-8, MMP-9] drive genesis and progression of lesions. Distinct lesions coexist such that inflammation in TB encompasses a spectrum of tissue changes. A better understanding of the multidimensionality of immunopathology in TB will inform novel therapies against this pulmonary disease.

Ridge Preservation Comparing a Nonresorbable PTFE Membrane to a Resorbable Collagen Membrane: A Clinical and Histologic Study in Humans.

Science.gov (United States)

Arbab, Hussain; Greenwell, Henry; Hill, Margaret; Morton, Dean; Vidal, Ricardo; Shumway, Brian; Allan, Nicholas D

2016-02-01

The primary aim of this randomized, controlled, blinded clinical trial was to compare the effect of a resorbable collagen membrane (CM group) versus a nonresorbable high-density polytetrafluoroethylene membrane (PTFE group) on the clinical and histologic outcomes of a ridge preservation procedure. All 24 sites received an intrasocket cancellous allograft and a buccal overlay bovine derived xenograft. The change in horizontal crestal ridge width was -1.4 ± 1.2 mm for the CM group, whereas the PTFE group lost -2.2 ± 1.5 mm, which was not statistically significant between groups (P > 0.05). Vertical ridge height change was -1.2 ± 1.5 for the CM group, whereas the PTFE group lost -0.5 ± 1.6, which was not significantly different between groups (P > 0.05). The percent vital bone was similar and not significantly different between groups. Primary closure was not obtained and the exposed membrane portion over the socket opening healed with keratinized tissue. The choice of a resorbable versus a nonresorbable barrier membrane did not affect the clinical or the histologic outcome of ridge preservation treatment.

Versatile High-Performance Regenerated Cellulose Membranes Prepared using Trimethylsilyl Cellulose as a Precursor

KAUST Repository

Puspasari, Tiara

2018-05-01

Cellulose has emerged as an indispensable membrane material due to its abundant availability, low cost, fascinating physiochemical properties and environment benignancy. However, it is believed that the potential of this polymer is not fully explored yet due to its insolubility in the common organic solvents, encouraging the use of derivatization-regeneration method as a viable alternative to the direct dissolution in exotic or reactive solvents. In this work, we use trimethylsilyl cellulose (TMSC), a highly soluble cellulose derivative, as a precursor for the fabrication of cellulose thin film composite membranes. TMSC is an attractive precursor to assemble thin cellulose films with good deposition behavior and film morphology; cumbersome solvents used in the one step cellulose processing are avoided. This derivative is prepared from cellulose by the known silylation reaction. The complete transformation of TMSC back into cellulose after the membrane formation is carried out by vapor-phase acid treatment, which is simple, scalable and reproducible. This process along with the initial TMSC concentration determines the membrane sieving characteristics. Unlike the typical regenerated cellulose membranes with meso- or macropores, membranes regenerated from TMSC display micropores suitable for the selective separation of nanomolecules in aqueous and organic solvent nanofiltration. The membranes introduced in this thesis represent the first polymeric membranes ever reported for highly selective separation of similarly sized small organic molecules based on charge and size differences with outstanding fluxes. Owing to its strong hydrophilic and amorphous character, the membranes also demonstrate excellent air-dehumidification performance as compared to previously reported thin film composite membranes. Moreover, the use of TMSC enables the creation of the previously unfeasible cellulose–polydimethylsiloxane (PDMS) and cellulose–polyethyleneimine (PEI) blend membranes

Designing block copolymer architectures for targeted membrane performance

KAUST Repository

Dorin, Rachel Mika

2014-01-01

Using a combination of block copolymer self-assembly and non-solvent induced phase separation, isoporous ultrafiltration membranes were fabricated from four poly(isoprene-b-styrene-b-4-vinylpyridine) triblock terpolymers with similar block volume fractions but varying in total molar mass from 43 kg/mol to 115 kg/mol to systematically study the effect of polymer size on membrane structure. Small-angle X-ray scattering was used to probe terpolymer solution structure in the dope. All four triblocks displayed solution scattering patterns consistent with a body-centered cubic morphology. After membrane formation, structures were characterized using a combination of scanning electron microscopy and filtration performance tests. Membrane pore densities that ranged from 4.53 × 1014 to 1.48 × 1015 pores/m 2 were observed, which are the highest pore densities yet reported for membranes using self-assembly and non-solvent induced phase separation. Hydraulic permeabilities ranging from 24 to 850 L m-2 h-1 bar-1 and pore diameters ranging from 7 to 36 nm were determined from permeation and rejection experiments. Both the hydraulic permeability and pore size increased with increasing molar mass of the parent terpolymer. The combination of polymer characterization and membrane transport tests described here demonstrates the ability to rationally design macromolecular structures to target specific performance characteristics in block copolymer derived ultrafiltration membranes. © 2013 Elsevier Ltd. All rights reserved.

Potential roles of cell-derived microparticles in ischemic brain disease.

Science.gov (United States)

Horstman, Lawrence L; Jy, Wenche; Bidot, Carlos J; Nordberg, Mary L; Minagar, Alireza; Alexander, J Steven; Kelley, Roger E; Ahn, Yeon S

2009-10-01

The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation. Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions. If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

Integral membrane protein structure determination using pseudocontact shifts

Energy Technology Data Exchange (ETDEWEB)

Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

2015-04-15

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

Membranes, methods of making membranes, and methods of separating gases using membranes

Science.gov (United States)

Ho, W. S. Winston

2012-10-02

Membranes, methods of making membranes, and methods of separating gases using membranes are provided. The membranes can include at least one hydrophilic polymer, at least one cross-linking agent, at least one base, and at least one amino compound. The methods of separating gases using membranes can include contacting a gas stream containing at least one of CO.sub.2, H.sub.2S, and HCl with one side of a nonporous and at least one of CO.sub.2, H.sub.2S, and HCl selectively permeable membrane such that at least one of CO.sub.2, H.sub.2S, and HCl is selectively transported through the membrane.

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Science.gov (United States)

Schäfer, Balázs; Orbán, Erika; Kele, Zoltán; Tömböly, Csaba

2015-01-01

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions. Copyright © 2015 John Wiley & Sons, Ltd.

Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

DEFF Research Database (Denmark)

Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel

2017-01-01

Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived...... from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains......, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3...

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

Science.gov (United States)

Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A; Kruse, Andrew C; Nurva, Shailika; Loland, Claus J; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H

2010-12-01

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons

Directory of Open Access Journals (Sweden)

Brian J. Wainger

2014-04-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1, C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1+/+ stem cell line do not display the hyperexcitability phenotype. SOD1A4V/+ ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.

Marine-derived collagen biomaterials from echinoderm connective tissues

KAUST Repository

Ferrario, Cinzia; Leggio, Livio; Leone, Roberta; Di Benedetto, Cristiano; Guidetti, Luca; Coccè , Valentina; Ascagni, Miriam; Bonasoro, Francesco; La Porta, Caterina A.M.; Candia Carnevali, M. Daniela; Sugni, Michela

2016-01-01

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

Marine-derived collagen biomaterials from echinoderm connective tissues

KAUST Repository

Ferrario, Cinzia

2016-03-31

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

Cost-Effective Method for Producing Self Supported Palladium Alloy Membranes for Use in Efficient Production of Coal Derived Hydrogen

Energy Technology Data Exchange (ETDEWEB)

K. Coulter

2008-03-31

Southwest Research Institute{reg_sign} (SwRI{reg_sign}) has utilized its expertise in large-area vacuum deposition methods to conduct research into the fabrication of dense, freestanding Pd-alloy membranes that are 3-5 microns thick and over 100 in{sup 2} in area. The membranes were deposited onto flexible and rigid supports that were subsequently removed and separated using novel techniques developed over the course of the project. Using these methods, the production of novel alloy compositions centered around the Pd-Cu system were developed with the objective of producing a thermally stable, nano-crystalline grain structure with the highest flux recorded as 242 SCFH/ft{sup 2} for a 2 {micro}m thick Pd{sub 53}Cu{sub 47} at 400 C and 20 psig feed pressure which when extrapolated is over twice the 2010 Department of Energy pure H{sub 2} flux target. Several membranes were made with the same permeability, but with different thicknesses and these membranes were highly selective. Researchers at the Colorado School of Mines supported the effort with extensive testing of experimental membranes as well as design and modeling of novel alloy composite structures. IdaTech provided commercial bench testing and analysis of SwRI-manufactured membranes. The completed deliverables for the project include test data on the performance of experimental membranes fabricated by vacuum deposition and several Pd-alloy membranes that were supplied to IdaTech for testing.

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

Science.gov (United States)

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Application of dynamic membranes in anaerobic membranes in anaerobic membrane bioreactor systems

NARCIS (Netherlands)

Erşahin, M.E.

2015-01-01

Anaerobic membrane bioreactors (AnMBRs) physically ensure biomass retention by the application of a membrane filtration process. With growing application experiences from aerobic membrane bioreactors (MBRs), the combination of membrane and anaerobic processes has received much attention and become

Reduced Graphene Oxide Membranes: Applications in Fog Collection and Water Purification

KAUST Repository

Tang, Bo

2017-05-01

Reduced graphene oxide (rGO) has attracted considerable interest recently as the low cost and chemical stable derivative of pristine graphene with application in many applications such as energy storage, water purification and electronic devices. This dissertation thoroughly investigated stacked rGO membrane fabrication process by vacuum-driven filtration, discovered asymmetry of the two surfaces of the rGO membrane, explored application perspectives of the asymmetric rGO membrane in fog collection and microstructure patterning, and disclosed membrane compaction issue during water filtration and species rejection. In more details, this dissertation revealed that, with suitable pore size, the filtration membrane substrate would leave its physical imprint on the bottom surface of the rGO membrane in the form of surface microstructures, which result in asymmetric dynamic water wettability properties of the two surfaces of the rGO membrane. The asymmetric wettability of the rGO membrane would lead to contrasting fog harvesting behavior of its two surfaces. The physical imprint mechanism was further extended to engineering pre-designed patterns selectively on the bottom surface of the rGO membrane. This dissertation, for the first time, reported the water flux and rejection kinetics, which was related to the compaction of the rGO membrane under pressure in the process of water filtration.

Deashing of coal liquids with ceramic membrane microfiltration and diafiltration

Energy Technology Data Exchange (ETDEWEB)

Bishop, B.; Goldsmith, R. [CeraMem Corp., Waltham, MA (United States)

1995-12-31

Removal of mineral matter from liquid hydrocarbons derived from the direct liquefaction of coal is required for product acceptability. Current methods include critical solvent deashing (Rose{sup {reg_sign}} process from Kerr-McGee) and filtration (U.S. Filter leaf filter as used by British Coal). These methods produce ash reject streams containing up to 15% of the liquid hydrocarbon product. Consequently, CeraMem proposed the use of low cost, ceramic crossflow membranes for the filtration of coal liquids bottoms to remove mineral matter and subsequent diafiltration (analogous to cake washing in dead-ended filtration) for the removal of coal liquid from the solids stream. The use of these ceramic crossflow membranes overcomes the limitations of traditional polymeric crossflow membranes by having the ability to operate at elevated temperature and to withstand prolonged exposure to hydrocarbon and solvent media. In addition, CeraMem`s membrane filters are significantly less expensive than competitive ceramic membranes due to their unique construction. With these ceramic membrane filters, it may be possible to reduce the product losses associated with traditional deashing processes at an economically attractive cost. The performance of these ceramic membrane microfilters is discussed.

Aluminum uptake from natural waters by a radiation-grafted membrane

Energy Technology Data Exchange (ETDEWEB)

Bazante-Yamaguishi, Renata; Moura, Eduardo; Manzoli, Jose E.; Geraldo, Aurea B.C., E-mail: ageraldo@ipen.br, E-mail: ryamaguishi@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2013-07-01

Styrene grafted, chemically modified polymeric membranes were used to carry off aluminum of drinking water from wells located at Billings dam region. The membranes comprised polymeric substrates of PVC (polyvinylchloride) and PP (polypropylene), which were mutually grafted with gamma radiation. The chemical modification included three basic reaction paths: Friedel-Crafts acylation, 2-methylanisole coupling and a final oxidation; this modification enables aluminum selectivity on the membrane. This chemical process inserts a salicylated derivative bonded onto the aromatic ring of styrene; such molecular arrangement is responsible for complexation of aluminum ions. The aluminum sorption capacity of these membranes was evaluated firstly from an aluminum control solution, where parameters like the ideal pH value for aluminum sorption and the interfering species were studied and correlated to know the best conditions for aluminum uptake. Later, the membranes were used for aluminum remediation of natural waters (real-life samples). The applicability results and limits are then discussed. (author)

Aluminum uptake from natural waters by a radiation-grafted membrane

International Nuclear Information System (INIS)

Bazante-Yamaguishi, Renata; Moura, Eduardo; Manzoli, Jose E.; Geraldo, Aurea B.C.

2013-01-01

Styrene grafted, chemically modified polymeric membranes were used to carry off aluminum of drinking water from wells located at Billings dam region. The membranes comprised polymeric substrates of PVC (polyvinylchloride) and PP (polypropylene), which were mutually grafted with gamma radiation. The chemical modification included three basic reaction paths: Friedel-Crafts acylation, 2-methylanisole coupling and a final oxidation; this modification enables aluminum selectivity on the membrane. This chemical process inserts a salicylated derivative bonded onto the aromatic ring of styrene; such molecular arrangement is responsible for complexation of aluminum ions. The aluminum sorption capacity of these membranes was evaluated firstly from an aluminum control solution, where parameters like the ideal pH value for aluminum sorption and the interfering species were studied and correlated to know the best conditions for aluminum uptake. Later, the membranes were used for aluminum remediation of natural waters (real-life samples). The applicability results and limits are then discussed. (author)

Currents and radiation from the large D black hole membrane

Energy Technology Data Exchange (ETDEWEB)

Bhattacharyya, Sayantani; Mandal, Anup Kumar [Department of Physics, Indian Institute of Technology Kanpur,Kalyanpur, Kanpur, Uttar Pradesh 208016 (India); Mandlik, Mangesh [Department of Theoretical Physics, Tata Institute of Fundamental Research,1, Homi Bhabha Road, Colaba, Mumbai, Maharashtra 400005 (India); Mehta, Umang [Department of Physics, Indian Institute of Technology Bombay,JVLR, Mumbai, Maharashtra 400076 (India); Minwalla, Shiraz [Department of Theoretical Physics, Tata Institute of Fundamental Research,1, Homi Bhabha Road, Colaba, Mumbai, Maharashtra 400005 (India); Sharma, Utkarsh [Department of Electrical Engineering, Indian Institute of Technology Bombay,JVLR, Mumbai, Maharashtra 400076 (India); Thakur, Somyadip [Department of Theoretical Physics, Tata Institute of Fundamental Research,1, Homi Bhabha Road, Colaba, Mumbai, Maharashtra 400005 (India)

2017-05-18

It has recently been demonstrated that black hole dynamics in a large number of dimensions D reduces to the dynamics of a codimension one membrane propagating in flat space. In this paper we define a stress tensor and charge current on this membrane and explicitly determine these currents at low orders in the expansion in (1/D). We demonstrate that dynamical membrane equations of motion derived in earlier work are simply conservation equations for our stress tensor and charge current. Through the paper we focus on solutions of the membrane equations which vary on a time scale of order unity. Even though the charge current and stress tensor are not parametrically small in such solutions, we show that the radiation sourced by the corresponding membrane currents is generically of order (1/(D{sup D})). In this regime it follows that the ‘near horizon’ membrane degrees of freedom are decoupled from asymptotic flat space at every perturbative order in the (1/D) expansion. We also define an entropy current on the membrane and use the Hawking area theorem to demonstrate that the divergence of the entropy current is point wise non negative. We view this result as a local form of the second law of thermodynamics for membrane motion.

Monitoring of praseodymium(III) ions in aqueous solutions, soil and sediment samples by a PVC membrane sensor based on a furan-triazole derivative

Energy Technology Data Exchange (ETDEWEB)

Pourjavid, Mohammad Reza [Islamic Azad University, Tehran (Iran, Islamic Republic of). Dept. of Medicinal Chemistry. Nuclear Science and Technology Research Institute; Rezaee, Mohammad; Hosseini, Majid Haji [Nuclear Science and Technology Research Institute, Tehran (Iran, Islamic Republic of); Razavi, Taherehsadat, E-mail: pourjavid@gmail.com [Research Institute of Petroleum Industry, Tehran (Iran, Islamic Republic of). Instrumental Analysis Dept.

2012-07-01

A furan-triazole derivative has been explored as an ionophore for preparation of a highly selective Pr(III) membrane sensor. The proposed sensor exhibits a Nernstian response for Pr(III) activity over a wide concentration range with a detection limit of 5.2 x 10{sup -8}M. Its response is independent of pH of the solution in the range 3.0-8.8 and offers the advantages of fast response time. To investigate the analytical applicability of the sensor, it was applied successfully as an indicator electrode in potentiometric titration of Pr(III) solution and also in the direct and indirect determination of trace Pr(III) ions in some samples. (author)

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers*

Science.gov (United States)

Antonenko, Yuri N.; Avetisyan, Armine V.; Cherepanov, Dmitry A.; Knorre, Dmitry A.; Korshunova, Galina A.; Markova, Olga V.; Ojovan, Silvia M.; Perevoshchikova, Irina V.; Pustovidko, Antonina V.; Rokitskaya, Tatyana I.; Severina, Inna I.; Simonyan, Ruben A.; Smirnova, Ekaterina A.; Sobko, Alexander A.; Sumbatyan, Natalia V.; Severin, Fedor F.; Skulachev, Vladimir P.

2011-01-01

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease. PMID:21454507

Origin and development of plasma membrane derived invaginations in Vinca rosea l.

Science.gov (United States)

Mahlberg, P.; Walkinshaw, C.; Olson, K.

1971-01-01

The occurrence, morphology, and possible ontogeny of plasma-membrane-related structures are described which can develop into invaginations or intravacuolar formations. An underlying study of meristematic tissues from the shoot of Vinca rosea supports the interpretation that endocytosis does occur in plant cells and that it is appropriate to refer to these structures as endocytoses. The function of these invaginations or their content remains to be elucidated.

Black holes: the membrane paradigm

International Nuclear Information System (INIS)

Thorne, K.S.; Price, R.H.; Macdonald, D.A.

1986-01-01

The physics of black holes is explored in terms of a membrane paradigm which treats the event horizon as a two-dimensional membrane embedded in three-dimensional space. A 3+1 formalism is used to split Schwarzschild space-time and the laws of physics outside a nonrotating hole, which permits treatment of the atmosphere in terms of the physical properties of thin slices. The model is applied to perturbed slowly or rapidly rotating and nonrotating holes, and to quantify the electric and magnetic fields and eddy currents passing through a membrane surface which represents a stretched horizon. Features of tidal gravitational fields in the vicinity of the horizon, quasars and active galalctic nuclei, the alignment of jets perpendicular to accretion disks, and the effects of black holes at the center of ellipsoidal star clusters are investigated. Attention is also given to a black hole in a binary system and the interactions of black holes with matter that is either near or very far from the event horizon. Finally, a statistical mechanics treatment is used to derive a second law of thermodynamics for a perfectly thermal atmosphere of a black hole

Polycyclic Aromatic Hydrocarbons as Plausible Prebiotic Membrane Components

Science.gov (United States)

Groen, Joost; Deamer, David W.; Kros, Alexander; Ehrenfreund, Pascale

2012-08-01

Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C6-C10 fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem

2017-10-12

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and/or impedance sensors) mounted on the porous surface. In another example, a membrane distillation (MD) process includes the membrane. Processing circuitry can be configured to monitor outputs of the plurality of sensors. The monitored outputs can be used to determine membrane degradation, membrane fouling, or to provide an indication of membrane replacement or cleaning. The sensors can also provide temperatures or temperature differentials across the porous surface, which can be used to improve modeling or control the MD process.

Manipulation of the Host Cell Membrane during Plasmodium Liver Stage Egress

Directory of Open Access Journals (Sweden)

Paul-Christian Burda

2017-04-01

Full Text Available A crucial step in the life cycle of Plasmodium parasites is the transition from the liver stage to the blood stage. Hepatocyte-derived merozoites reach the blood vessels of the liver inside host cell-derived vesicles called merosomes. The molecular basis of merosome formation is only partially understood. Here we show that Plasmodium berghei liver stage merozoites, upon rupture of the parasitophorous vacuole membrane, destabilize the host cell membrane (HCM and induce separation of the host cell actin cytoskeleton from the HCM. At the same time, the phospholipid and protein composition of the HCM appears to be substantially altered. This includes the loss of a phosphatidylinositol 4,5-bisphosphate (PIP2 reporter and the PIP2-dependent actin-plasma membrane linker ezrin from the HCM. Furthermore, transmembrane domain-containing proteins and palmitoylated and myristoylated proteins, as well as glycosylphosphatidylinositol-anchored proteins, lose their HCM localization. Collectively, these findings provide an explanation of HCM destabilization during Plasmodium liver stage egress and thereby contribute to our understanding of the molecular mechanisms that lead to merosome formation.

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.; Choi, Seung Hak

2012-01-01

. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane

Recent advances on polymeric membranes for membrane reactors

KAUST Repository

Buonomenna, M. G.

2012-06-24

Membrane reactors are generally applied in high temperature reactions (>400 °C). In the field of fine chemical synthesis, however, much milder conditions are generally applicable and polymeric membranes were applied without their damage. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane is to be used in. In this chapter a review of up to date literature about polymers and configuration catalyst/ membranes used in some recent polymeric membrane reactors is given. The new emerging concept of polymeric microcapsules as catalytic microreactors has been proposed. © 2012 Bentham Science Publishers. All rights reserved.

Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor.

Science.gov (United States)

Shishkanova, T V; Volf, R; Krondak, M; Král, V

2007-05-15

A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

Science.gov (United States)

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Biogenesis of the demarcation membrane system (DMS) in megakaryocytes.

Science.gov (United States)

Eckly, Anita; Heijnen, Harry; Pertuy, Fabien; Geerts, Willie; Proamer, Fabienne; Rinckel, Jean-Yves; Léon, Catherine; Lanza, François; Gachet, Christian

2014-02-06

The demarcation membrane system (DMS) in megakaryocytes forms the plasma membrane (PM) of future platelets. Using confocal microscopy, electron tomography, and large volume focused ion beam/scanning electron microscopy (FIB/SEM), we determined the sequential steps of DMS formation. We identified a pre-DMS that initiated at the cell periphery and was precisely located between the nuclear lobes. At all developmental stages, the DMS remained continuous with the cell surface. The number of these connections correlated well with the nuclear lobulation, suggesting a relationship with cleavage furrow formation and abortive cytokinesis. On DMS expansion, Golgi complexes assembled around the pre-DMS, and fusion profiles between trans-golgi network-derived vesicles and the DMS were observed. Brefeldin-A reduced DMS expansion, indicating that the exocytic pathway is essential for DMS biogenesis. Close contacts between the endoplasmic reticulum (ER) and the DMS were detected, suggesting physical interaction between the 2 membrane systems. FIB/SEM revealed that the DMS forms an intertwined tubular membrane network resembling the platelet open canalicular system. We thus propose the following steps in DMS biogenesis: (1) focal membrane assembly at the cell periphery; (2) PM invagination and formation of a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and (4) ER-mediated lipid transfer.

Biofouling in capillary and spiral wound membranes facilitated by marine algal bloom

KAUST Repository

Villacorte, L.O.

2017-10-11

Algal-derived organic matter (AOM), particularly transparent exopolymer particles, has been suspected to facilitate biofilm development in membrane systems (e.g., seawater reverse osmosis). This study demonstrates the possible role of AOM on biofouling in membrane systems affected by marine algal blooms. The tendency of AOM from bloom-forming marine algae to adhere to membranes and its ability to enhance biofilm growth were measured using atomic force microscopy, flow cytometry, liquid chromatography and accelerated membrane biofouling experiments. Adhesion force measurements indicate that AOM tends to adhere to clean membranes and even more strongly to AOM-fouled membranes. Batch growth tests illustrate that the capacity of seawater to support bacterial growth can significantly increase with AOM concentration. Biofouling experiments with spiral wound and capillary membranes illustrate that when nutrients availability are not limited in the feed water, a high concentration of AOM – whether in suspension or attached to the membrane – can substantially accelerates biofouling. A significantly lower biofouling rate was observed on membranes exposed to feed water spiked only with AOM or easily biodegradable nutrients. The abovementioned findings indicate that AOM facilitates the onset of membrane biofouling primarily as a conditioning platform and to some extent as a nutrient source for biofilm-forming bacteria.

Biofouling in capillary and spiral wound membranes facilitated by marine algal bloom

KAUST Repository

Villacorte, L.O.; Ekowati, Y.; Calix-Ponce, H.N.; Kisielius, V.; Kleijn, J.M.; Vrouwenvelder, Johannes S.; Schippers, J.C.; Kennedy, M.D.

2017-01-01

Algal-derived organic matter (AOM), particularly transparent exopolymer particles, has been suspected to facilitate biofilm development in membrane systems (e.g., seawater reverse osmosis). This study demonstrates the possible role of AOM on biofouling in membrane systems affected by marine algal blooms. The tendency of AOM from bloom-forming marine algae to adhere to membranes and its ability to enhance biofilm growth were measured using atomic force microscopy, flow cytometry, liquid chromatography and accelerated membrane biofouling experiments. Adhesion force measurements indicate that AOM tends to adhere to clean membranes and even more strongly to AOM-fouled membranes. Batch growth tests illustrate that the capacity of seawater to support bacterial growth can significantly increase with AOM concentration. Biofouling experiments with spiral wound and capillary membranes illustrate that when nutrients availability are not limited in the feed water, a high concentration of AOM – whether in suspension or attached to the membrane – can substantially accelerates biofouling. A significantly lower biofouling rate was observed on membranes exposed to feed water spiked only with AOM or easily biodegradable nutrients. The abovementioned findings indicate that AOM facilitates the onset of membrane biofouling primarily as a conditioning platform and to some extent as a nutrient source for biofilm-forming bacteria.

Membranes: A Variety of Energy Landscapes for Many Transfer Opportunities.

Science.gov (United States)

Bacchin, Patrice

2018-02-22

A membrane can be represented by an energy landscape that solutes or colloids must cross. A model accounting for the momentum and the mass balances in the membrane energy landscape establishes a new way of writing for the Darcy law. The counter-pressure in the Darcy law is no longer written as the result of an osmotic pressure difference but rather as a function of colloid-membrane interactions. The ability of the model to describe the physics of the filtration is discussed in detail. This model is solved in a simplified energy landscape to derive analytical relationships that describe the selectivity and the counter-pressure from ab initio operating conditions. The model shows that the stiffness of the energy landscape has an impact on the process efficiency: a gradual increase in interactions (such as with hourglass pore shape) can reduce the separation energetic cost. It allows the introduction of a new paradigm to increase membrane efficiency: the accumulation that is inherent to the separation must be distributed across the membrane. Asymmetric interactions thus lead to direction-dependent transfer properties and the membrane exhibits diode behavior. These new transfer opportunities are discussed.

Descriptions of membrane mechanics from microscopic and effective two-dimensional perspectives

International Nuclear Information System (INIS)

Lomholt, Michael A; Miao Ling

2006-01-01

Mechanics of fluid membranes may be described in terms of the concepts of mechanical deformations and stresses or in terms of mechanical free-energy functions. In this paper, each of the two descriptions is developed by viewing a membrane from two perspectives: a microscopic perspective, in which the membrane appears as a thin layer of finite thickness and with highly inhomogeneous material and force distributions in its transverse direction, and an effective, two-dimensional perspective, in which the membrane is treated as an infinitely thin surface, with effective material and mechanical properties. A connection between these two perspectives is then established. Moreover, the functional dependence of the variation in the mechanical free energy of the membrane on its mechanical deformations is first studied in the microscopic perspective. The result is then used to examine to what extent different, effective mechanical stresses and forces can be derived from a given, effective functional of the mechanical free energy

The large D black hole membrane paradigm at first subleading order

International Nuclear Information System (INIS)

Dandekar, Yogesh; De, Anandita; Mazumdar, Subhajit; Minwalla, Shiraz; Saha, Arunabha

2016-01-01

In the large D limit, and under certain circumstances, it has recently been demonstrated that black hole dynamics in asymptotically flat spacetime reduces to the dynamics of a non gravitational membrane propagating in flat D dimensional spacetime. We demonstrate that this correspondence extends to all orders in a 1/D expansion and outline a systematic method for deriving the corrected membrane equation in a power series expansion in 1/D. As an illustration of our method we determine the first subleading corrections to the membrane equations of motion. A qualitatively new effect at this order is that the divergence of the membrane velocity is nonzero and proportional to the square of the shear tensor reminiscent of the entropy current of hydrodynamics. As a test, we use our modified membrane equations to compute the corrections to frequencies of light quasinormal modes about the Schwarzschild black hole and find a perfect match with earlier computations performed directly in the gravitational bulk.

The large D black hole membrane paradigm at first subleading order

Energy Technology Data Exchange (ETDEWEB)

Dandekar, Yogesh [Department of Theoretical Physics, Tata Institute of Fundamental Research,Homi Bhabha Road, Mumbai 400005 (India); De, Anandita [Department of Physics, Indian Institute of Science Education and Research,Dr. Homi Bhabha Road, Pashan, Pune 411008 (India); Mazumdar, Subhajit; Minwalla, Shiraz; Saha, Arunabha [Department of Theoretical Physics, Tata Institute of Fundamental Research,Homi Bhabha Road, Mumbai 400005 (India)

2016-12-21

In the large D limit, and under certain circumstances, it has recently been demonstrated that black hole dynamics in asymptotically flat spacetime reduces to the dynamics of a non gravitational membrane propagating in flat D dimensional spacetime. We demonstrate that this correspondence extends to all orders in a 1/D expansion and outline a systematic method for deriving the corrected membrane equation in a power series expansion in 1/D. As an illustration of our method we determine the first subleading corrections to the membrane equations of motion. A qualitatively new effect at this order is that the divergence of the membrane velocity is nonzero and proportional to the square of the shear tensor reminiscent of the entropy current of hydrodynamics. As a test, we use our modified membrane equations to compute the corrections to frequencies of light quasinormal modes about the Schwarzschild black hole and find a perfect match with earlier computations performed directly in the gravitational bulk.

Sensing Phosphatidylserine in Cellular Membranes

Directory of Open Access Journals (Sweden)

Jason G. Kay

2011-01-01

Full Text Available Phosphatidylserine, a phospholipid with a negatively charged head-group, is an important constituent of eukaryotic cellular membranes. On the plasma membrane, rather than being evenly distributed, phosphatidylserine is found preferentially in the inner leaflet. Disruption of this asymmetry, leading to the appearance of phosphatidylserine on the surface of the cell, is known to play a central role in both apoptosis and blood clotting. Despite its importance, comparatively little is known about phosphatidylserine in cells: its precise subcellular localization, transmembrane topology and intracellular dynamics are poorly characterized. The recent development of new, genetically-encoded probes able to detect phosphatidylserine within live cells, however, is leading to a more in-depth understanding of the biology of this phospholipid. This review aims to give an overview of the current methods for phosphatidylserine detection within cells, and some of the recent realizations derived from their use.

Killing of melanoma cells and their metastases by human lactoferricin derivatives requires interaction with the cancer marker phosphatidylserine.

Science.gov (United States)

Riedl, Sabrina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2014-10-01

Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.

Modeling of interactions between nanoparticles and cell membranes

Science.gov (United States)

Ban, Young-Min

Rapid development of nanotechnology and ability to manufacture materials and devices with nanometer feature size leads to exciting innovations in many areas including the medical and electronic fields. However, the possible health and environmental impacts of manufactured nanomaterials are not fully known. Recent experimental reports suggest that some of the manufactured nanomaterials, such as fullerenes and carbon nanotubes, are highly toxic even in small concentrations. The goal of the current work is to understand the mechanisms responsible for the toxicity of nanomaterials. In the current study coarse-grained molecular dynamics simulations are employed to investigate the interactions between NPs and cellular membranes at a molecular level. One of the possible toxicity mechanisms of the nanomaterials is membrane disruption. Possibility of membrane disruption exposed to the manufactured nanomaterials are examined by considering chemical reactions and non-reactive physical interactions as chemical as well as physical mechanisms. Mechanisms of transport of carbon-based nanoparticles (fullerene and its derivative) across a phospholipid bilayer are investigated. The free energy profile is obtained using constrained simulations. It is shown that the considered nanoparticles are hydrophobic and therefore they tend to reside in the interior of the lipid bilayer. In addition, the dynamics of the membrane fluctuations is significantly affected by the nanoparticles at the bilayer-water interface. The hydrophobic interaction between the particles and membrane core induces the strong coupling between the nanoparticle motion and membrane deformation. It is observed that the considered nanoparticles affect several physical properties of the membrane. The nanoparticles embedded into the membrane interior lead to the membrane softening, which becomes more significant with increase in CNT length and concentration. The lateral pressure profile and membrane energy in the membrane

Membrane fouling mechanism of biofilm-membrane bioreactor (BF-MBR): Pore blocking model and membrane cleaning.

Science.gov (United States)

Zheng, Yi; Zhang, Wenxiang; Tang, Bing; Ding, Jie; Zheng, Yi; Zhang, Zhien

2018-02-01

Biofilm membrane bioreactor (BF-MBR) is considered as an important wastewater treatment technology that incorporates advantages of both biofilm and MBR process, as well as can alleviate membrane fouling, with respect to the conventional activated sludge MBR. But, to be efficient, it necessitates the establishment of proper methods for the assessment of membrane fouling. Four Hermia membrane blocking models were adopted to quantify and evaluate the membrane fouling of BF-MBR. The experiments were conducted with various operational conditions, including membrane types, agitation speeds and transmembrane pressure (TMP). Good agreement between cake formation model and experimental data was found, confirming the validity of the Hermia models for assessing the membrane fouling of BF-MBR and that cake layer deposits on membrane. Moreover, the influences of membrane types, agitation speeds and transmembrane pressure on the Hermia pore blocking coefficient of cake layer were investigated. In addition, the permeability recovery after membrane cleaning at various operational conditions was studied. This work confirms that, unlike conventional activated sludge MBR, BF-MBR possesses a low degree of membrane fouling and a higher membrane permeability recovery after cleaning. Copyright © 2017 Elsevier Ltd. All rights reserved.

Graphene oxide doped ionic liquid ultrathin composite membranes for efficient CO2 capture

KAUST Repository

Karunakaran, Madhavan

2016-11-28

Advanced membrane systems with high flux and sufficient selectivity are required for industrial gas separation processes. In order to achieve high flux and high selectivity, the membrane material should be as thin as possible and it should have selective sieving channels and long term stability. This could be achieved by designing a three component material consisting of a blend of an ionic liquid and graphene oxide covered by a highly permeable low selective polymeric coating. By using a simple dip coating technique, we prepared high flux and CO selective ultrathin graphene oxide (GO)/ionic liquid membranes on a porous ultrafiltration support. The ultrathin composite membranes derived from GO/ionic liquid complex displays remarkable combinations of permeability (CO flux: 37 GPU) and selectivity (CO/N selectivity: 130) that surpass the upper bound of ionic liquid membranes for CO/N separation. Moreover, the membranes were stable when tested for 120 hours.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

Science.gov (United States)

Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten

2016-09-01

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Transport of Eu3+ through a Bis(2-ethylhexyl)-phosphoric acid, n-dodecane solid supported liquid membrane

International Nuclear Information System (INIS)

Danesi, P.R.; Horwitz, E.P.; Rickert, P.

1982-01-01

The coupled transpot of Eu 3 + and H + ions througn a solid supported liquid membrane consisting of a porous polypropylene film immobilizing an HDEHP solution in n-dodecane has been studied as a function of the membrane area, stirring speed of the aqueous solutions, membrane composition, and acidity of the feed solution. The experimental results are in agreement with predictions derived from a theoretical permeability coefficient equation which assumes that membrane diffusion and aqueous film diffusion are the only rate-controlling factors

High temperature transport properties of polyphosphazene membranes for direct methanol fuel cells

Energy Technology Data Exchange (ETDEWEB)

Xiangyang Zhou; Chalkova, E. [Pennsylvania State University (United States). The Energy Institute; Weston, J.; Lvov, S.N. [Pennsylvania State University (United States). The Energy Institute; Pennsylvania State University (United States). Department of Energy and Geo-Environment Engineering; Hofmann, M.A.; Ambler, C.M.; Allcock, H.R. [Pennsylvania State University (United States). Department of Chemistry

2003-06-30

Experimental methods for studying the conductivity and methanol permeability of proton conductive polymers over a wide range of temperatures have been developed. The proton conductivity and methanol permeability of several polymer electrolyte membranes including sulfonated and phosphonated poly[(aryloxy)phosphazenes] was determined at temperatures up to 120 {sup o}C. Nafion 117 membranes were tested using the same methods in order to determine the reliability of the methods. Although the conductivities of the polyphosphazene membranes were either similar to or lower than that of the Nafion 117 membranes, they continue to hold promise for fuel cell applications. We observed similar activation energies of proton conduction for Nafion 117, and for sulfonated and phosphonated polyphosphazene membranes. However, the methanol permeability of a sulfonated membrane was about 8 times lower than that of the Nafion 117 membrane at room temperature although the values were comparable at 120 {sup o}C. The permeability of a phosphonated phosphazene derivative was about 40 times lower than that of the Nafion 117 membrane at room temperature and about 9 times lower at 120 {sup o}C. This is a significant improvement over the behavior of Nafion 117. (author)

High temperature transport properties of polyphosphazene membranes for direct methanol fuel cells

Energy Technology Data Exchange (ETDEWEB)

Zhou Xiangyang; Weston, Jamie; Chalkova, Elena; Hofmann, Michael A.; Ambler, Catherine M.; Allcock, Harry R.; Lvov, Serguei N

2003-06-30

Experimental methods for studying the conductivity and methanol permeability of proton conductive polymers over a wide range of temperatures have been developed. The proton conductivity and methanol permeability of several polymer electrolyte membranes including sulfonated and phosphonated poly[(aryloxy)phosphazenes] was determined at temperatures up to 120 deg. C. Nafion 117 membranes were tested using the same methods in order to determine the reliability of the methods. Although the conductivities of the polyphosphazene membranes were either similar to or lower than that of the Nafion 117 membranes, they continue to hold promise for fuel cell applications. We observed similar activation energies of proton conduction for Nafion 117, and for sulfonated and phosphonated polyphosphazene membranes. However, the methanol permeability of a sulfonated membrane was about 8 times lower than that of the Nafion 117 membrane at room temperature although the values were comparable at 120 deg. C. The permeability of a phosphonated phosphazene derivative was about 40 times lower than that of the Nafion 117 membrane at room temperature and about 9 times lower at 120 deg. C. This is a significant improvement over the behavior of Nafion 117.

High temperature transport properties of polyphosphazene membranes for direct methanol fuel cells

International Nuclear Information System (INIS)

Zhou Xiangyang; Weston, Jamie; Chalkova, Elena; Hofmann, Michael A.; Ambler, Catherine M.; Allcock, Harry R.; Lvov, Serguei N.

2003-01-01

Experimental methods for studying the conductivity and methanol permeability of proton conductive polymers over a wide range of temperatures have been developed. The proton conductivity and methanol permeability of several polymer electrolyte membranes including sulfonated and phosphonated poly[(aryloxy)phosphazenes] was determined at temperatures up to 120 deg. C. Nafion 117 membranes were tested using the same methods in order to determine the reliability of the methods. Although the conductivities of the polyphosphazene membranes were either similar to or lower than that of the Nafion 117 membranes, they continue to hold promise for fuel cell applications. We observed similar activation energies of proton conduction for Nafion 117, and for sulfonated and phosphonated polyphosphazene membranes. However, the methanol permeability of a sulfonated membrane was about 8 times lower than that of the Nafion 117 membrane at room temperature although the values were comparable at 120 deg. C. The permeability of a phosphonated phosphazene derivative was about 40 times lower than that of the Nafion 117 membrane at room temperature and about 9 times lower at 120 deg. C. This is a significant improvement over the behavior of Nafion 117

Adsorptive removal of phenolic compounds using cellulose acetate phthalate–alumina nanoparticle mixed matrix membrane

Energy Technology Data Exchange (ETDEWEB)

Mukherjee, Raka; De, Sirshendu, E-mail: sde@che.iitkgp.ernet.in

2014-01-30

Highlights: • Composite membrane of cellulose–acetate–phthalate and alumina nanoparticle is cast. • Surface charge of the membrane changes with nanoparticle concentration and pH. • Separation of phenolic compounds occurs due to adsorption. • The removal efficiency is maximum for 20% nanoparticle with 91% removal of catechol. • Transmembrane pressure drop has negligible effect on solute separation. -- Abstract: Mixed matrix membranes (MMMs) were prepared using alumina nanoparticles and cellulose acetate phthalate (CAP) by varying concentration of nanoparticles in the range of 10 to 25 wt%. The membranes were characterized by scanning electron micrograph, porosity, permeability, molecular weight cut off, contact angle, surface zeta potential, mechanical strength. Addition of nanoparticles increased the porosity, permeability of the membrane up to 20 wt% of alumina. pH at point of zero charge of the membrane was 5.4. Zeta potential of the membrane became more negative up to 20 wt% of nanoparticles. Adsorption of phenolic derivatives, catechol, paranitrophenol, phenol, orthochloro phenol, metanitrophenol, by MMMs were investigated. Variation of rejection and permeate flux profiles were studied for different solutes as a function of various operating conditions, namely, solution pH, solute concentration in feed and transmembrane pressure drop. Difference in rejection of phenolic derivatives is consequence of interplay of surface charge and adsorption by alumina. Adsorption isotherm was fitted for different solutes and effects of pH were investigated. Catechol showed the maximum rejection 91% at solution pH 9. Addition of electrolyte reduced the rejection of solutes. Transmembrane pressure drop has insignificant effects on solute rejection. Competitive adsorption reduced the rejection of individual solute.

Membrane paradigm and entropy of black holes in the Euclidean action approach

International Nuclear Information System (INIS)

Lemos, Jose P. S.; Zaslavskii, Oleg B.

2011-01-01

The membrane paradigm approach to black holes fixes in the vicinity of the event horizon a fictitious surface, the stretched horizon, so that the spacetime outside remains unchanged and the spacetime inside is vacuum. Using this powerful method, several black hole properties have been found and settled, such as the horizon's viscosity, electrical conductivity, resistivity, as well as other properties. On the other hand, the Euclidean action approach to black hole spacetimes has been very fruitful in understanding black hole entropy. Combining both the Euclidean action and membrane paradigm approaches, a direct derivation of the black hole entropy is given. In the derivation, it is considered that the only fields present are the gravitational and matter fields, with no electric field.

Morphological changes in vesicles and release of an encapsulated compound triggered by a photoresponsive Malachite Green leuconitrile derivative.

Science.gov (United States)

Uda, Ryoko M; Hiraishi, Eri; Ohnishi, Ryo; Nakahara, Yoshio; Kimura, Keiichi

2010-04-20

Photoinduced morphological changes in phosphatidylcholine vesicles are triggered by a Malachite Green leuconitrile derivative dissolved in the lipidic membrane, and are observed at Malachite Green derivative/lipid ratios Malachite Green derivative is a photoresponsive compound that undergoes ionization to afford a positive charge on the molecule by UV irradiation. The Malachite Green derivative exhibits amphiphilicity when ionized photochemically, whereas it behaves as a lipophilic compound under dark conditions. Cryo-transmission electron microscopy was used to determine vesicle morphology. The effects of the Malachite Green derivative on vesicles were studied by dynamic light scattering and fluorescence resonance energy transfer. Irradiation of vesicles containing the Malachite Green derivative induces nonspherical vesicle morphology, fusion of vesicles, and membrane solubilization, depending on conditions. Furthermore, irradiation of the Malachite Green derivative induces the release of a vesicle-encapsulated compound.

Correlated volume-energy fluctuations of phospholipid membranes: A simulation study

DEFF Research Database (Denmark)

Pedersen, Ulf. R.; Peters, Günther H.J.; Schröder, Thomas B.

2010-01-01

This paper reports all-atom computer simulations of five phospholipid membranes (DMPC, DPPC, DMPG, DMPS, and DMPSH) with focus on the thermal equilibrium fluctuations of volume, energy, area, thickness, and chain order. At constant temperature and pressure, volume and energy exhibit strong...... membranes, showing a similar picture. The cause of the observed strong correlations is identified by splitting volume and energy into contributions from tails, heads, and water, and showing that the slow volume−energy fluctuations derive from van der Waals interactions of the tail region; they are thus...

New insights into membrane-active action in plasma membrane of fungal hyphae by the lipopeptide antibiotic bacillomycin L.

Science.gov (United States)

Zhang, Bao; Dong, Chunjuan; Shang, Qingmao; Han, Yuzhu; Li, Pinglan

2013-09-01

Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activities against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. Prior to this study, the role of membrane permeabilization in the antimicrobial activity of bacillomycin L against plant pathogenic fungi had not been investigated. To shed light on the mechanism of this antifungal activity, the permeabilization of R. solani hyphae by bacillomycin L was investigated and compared with that by amphotericin B, a polyene antibiotic which is thought to act primarily through membrane disruption. Our results derived from electron microscopy, various fluorescent techniques and gel retardation experiments revealed that the antifungal activity of bacillomycin L may be not solely a consequence of fungal membrane permeabilization, but related to the interaction of it with intracellular targets. Our findings provide more insights into the mode of action of bacillomycin L and other iturins, which could in turn help to develop new or improved antifungal formulations or result in novel strategies to prevent fungal spoilage. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Membrane dynamics

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

Science.gov (United States)

Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

2015-08-01

Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carbon molecular sieve dense film membranes derived from Matrimid® for ethylene/ethane separation

KAUST Repository

Rungta, Meha; Xu, Liren; Koros, William J.

2012-01-01

Development of dense film carbon molecular sieve (CMS) membranes for ethylene/ethane (C 2H 4/C 2H 6) separation is reported. A commercial polyimide, Matrimid®, was pyrolyzed under vacuum and inert argon atmosphere, and the resultant CMS films were

Rate and mechanism of facilitated americium(III) transport through a supported liquid membrane containing a bifunctional organophosphorus mobile carrier

International Nuclear Information System (INIS)

Danesi, P.R.; Horwitz, E.P.; Rickert, P.G.

1983-01-01

The facilitated transport of Am(III) from aqueous nitrate solutions to formic acid aqueous solutions through a supported liquid membrane (SLM) is described. The supported liquid membrane consists of a solution of a new (carbamoylmethyl)phosphine oxide in diethylbenzene (DEB) absorbed into a 48 μm thick microporous polypropylene film. The transport mechanism consists of a diffusion process through an aqueous diffusion film, a fast interfacial chemical reaction, and diffusion through the membrane itself. Equations describing the rate of transport are derived. They correlate the membrane permeability coefficient to diffusional parameters and to the chemical composition of the system. Different rate-controlling processes are shown to control the membrane permeability when the composition of the system is varied and as long as the transport occurs. The experimental data are quantitatively explained with the derived equations. The diffusion coefficient of the permeating species and the equilibrium constant of the fast interfacial reactions are evaluated. 13 figures, 1 table

The fractal dimension of cell membrane correlates with its capacitance: A new fractal single-shell model

Science.gov (United States)

Wang, Xujing; Becker, Frederick F.; Gascoyne, Peter R. C.

2010-01-01

The scale-invariant property of the cytoplasmic membrane of biological cells is examined by applying the Minkowski–Bouligand method to digitized scanning electron microscopy images of the cell surface. The membrane is found to exhibit fractal behavior, and the derived fractal dimension gives a good description of its morphological complexity. Furthermore, we found that this fractal dimension correlates well with the specific membrane dielectric capacitance derived from the electrorotation measurements. Based on these findings, we propose a new fractal single-shell model to describe the dielectrics of mammalian cells, and compare it with the conventional single-shell model (SSM). We found that while both models fit with experimental data well, the new model is able to eliminate the discrepancy between the measured dielectric property of cells and that predicted by the SSM. PMID:21198103

Smart membranes for monitoring membrane based desalination processes

KAUST Repository

Laleg-Kirati, Taous-Meriem; Karam, Ayman M.

2017-01-01

Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and

Tides on Europa: The membrane paradigm

Science.gov (United States)

Beuthe, Mikael

2015-03-01

Jupiter's moon Europa has a thin icy crust which is decoupled from the mantle by a subsurface ocean. The crust thus responds to tidal forcing as a deformed membrane, cold at the top and near melting point at the bottom. In this paper I develop the membrane theory of viscoelastic shells with depth-dependent rheology with the dual goal of predicting tidal tectonics and computing tidal dissipation. Two parameters characterize the tidal response of the membrane: the effective Poisson's ratio ν bar and the membrane spring constant Λ, the latter being proportional to the crust thickness and effective shear modulus. I solve membrane theory in terms of tidal Love numbers, for which I derive analytical formulas depending on Λ, ν bar , the ocean-to-bulk density ratio and the number k2∘ representing the influence of the deep interior. Membrane formulas predict h2 and k2 with an accuracy of a few tenths of percent if the crust thickness is less than one hundred kilometers, whereas the error on l2 is a few percents. Benchmarking with the thick-shell software SatStress leads to the discovery of an error in the original, uncorrected version of the code that changes stress components by up to 40%. Regarding tectonics, I show that different stress-free states account for the conflicting predictions of thin and thick shell models about the magnitude of tensile stresses due to nonsynchronous rotation. Regarding dissipation, I prove that tidal heating in the crust is proportional to Im (Λ) and that it is equal to the global heat flow (proportional to Im (k2)) minus the core-mantle heat flow (proportional to Im (k2∘)). As an illustration, I compute the equilibrium thickness of a convecting crust. More generally, membrane formulas are useful in any application involving tidal Love numbers such as crust thickness estimates, despinning tectonics or true polar wander.

Biomimetic membranes and methods of making biomimetic membranes

Science.gov (United States)

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics

Science.gov (United States)

Han, Zhou; Jin, Lei; Platisa, Jelena; Cohen, Lawrence B.; Baker, Bradley J.; Pieribone, Vincent A.

2013-01-01

We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals. PMID:24312287

Fluorescent protein voltage probes derived from ArcLight that respond to membrane voltage changes with fast kinetics.

Directory of Open Access Journals (Sweden)

Zhou Han

Full Text Available We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ less than 6 ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9% is not as large as the Ciona-based ArcLight (~35%, they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.

Pilot Scale Water Gas Shift - Membrane Device for Hydrogen from Coal

Energy Technology Data Exchange (ETDEWEB)

Barton, Tom [Western Research Inst. (WRI), Laramie, WY (United States)

2013-09-01

The objectives of the project were to build pilot scale hydrogen separation systems for use in a gasification product stream. This device would demonstrate fabrication and manufacturing techniques for producing commercially ready facilities. The design was a 2 lb/day hydrogen device which included composite hydrogen separation membranes, a water gas shift monolith catalyst, and stainless steel structural components. Synkera Technologies was to prepare hydrogen separation membranes with metallic rims, and to adjust the alloy composition in their membranes to a palladium-gold composition which is sulfur resistant. Chart was to confirm their brazing technology for bonding the metallic rims of the composite membranes to their structural components and design and build the 2 lbs/day device incorporating membranes and catalysts. WRI prepared the catalysts and completed the testing of the membranes and devices on coal derived syngas. The reactor incorporated eighteen 2'' by 7'' composite palladium alloy membranes. These membranes were assembled with three stacks of three paired membranes. Initial vacuum testing and visual inspection indicated that some membranes were cracked, either in transportation or in testing. During replacement of the failed membranes, while pulling a vacuum on the back side of the membranes, folds were formed in the flexible composite membranes. In some instances these folds led to cracks, primarily at the interface between the alumina and the aluminum rim. The design of the 2 lb/day device was compromised by the lack of any membrane isolation. A leak in any membrane failed the entire device. A large number of tests were undertaken to bring the full 2 lb per day hydrogen capacity on line, but no single test lasted more than 48 hours. Subsequent tests to replace the mechanical seals with brazing have been promising, but the technology remains promising but not proven.

Gel layer formation on membranes in Membrane Bioreactors

NARCIS (Netherlands)

Van den Brink, P.F.H.

2014-01-01

The widespread application of membrane bioreactors (MBRs) for municipal wastewater treatment is hampered by membrane fouling. Fouling increases energy demand, reduces process performance and creates the need for more frequent (chemical) membrane cleaning or replacement. Membrane fouling in MBRs is

Nanodisc-solubilized membrane protein library reflects the membrane proteome.

Science.gov (United States)

Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

2013-05-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

Study of Membrane Reflector Technology

Science.gov (United States)

Knapp, K.; Hedgepeth, J.

1979-01-01

Very large reflective surfaces are required by future spacecraft for such purposes as solar energy collection, antenna surfaces, thermal control, attitude and orbit control with solar pressure, and solar sailing. The performance benefits in large membrane reflector systems, which may be derived from an advancement of this film and related structures technology, are identified and qualified. The results of the study are reported and summarized. Detailed technical discussions of various aspects of the study are included in several separate technical notes which are referenced.

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

Science.gov (United States)

Orozco, Aaron F.; Jorgez, Carolina J.; Horne, Cassandra; Marquez-Do, Deborah A.; Chapman, Matthew R.; Rodgers, John R.; Bischoff, Farideh Z.; Lewis, Dorothy E.

2008-01-01

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. PMID:18974299

Crystal structure of the plasma membrane proton pump

DEFF Research Database (Denmark)

Pedersen, Bjørn P.; Buch-Pedersen, Morten Jeppe; Morth, J. Preben

2007-01-01

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H1-ATPase (the proton pump) in plants and fungi1......-3, and Na1,K1-ATPase (the sodium-potassium pump) in animals4. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis5.The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na1,K1-ATPase and Ca21......- ATPase are type II6. Electron microscopy has revealed the overall shape of proton pumps7, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define...

Studies on membrane acid electrolysis for hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Silva, Marco Antonio Oliveira da; Linardi, Marcelo; Saliba-Silva, Adonis Marcelo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Centro de Celulas a Combustivel e Hidrogenio], Email: saliba@ipen.br

2010-07-01

Hydrogen represents great opportunity to be a substitute for fossil fuels in the future. Water as a renewable source of hydrogen is of great interest, since it is abundant and can decompose, producing only pure H{sub 2} and O{sub 2}. This decomposition of water can be accomplished by processes such as electrolysis, thermal decomposition and thermochemical cycles. The electrolysis by membrane has been proposed as a viable process for hydrogen production using thermal and electrical energy derived from nuclear energy or any renewable source like solar energy. In this work, within the context of optimization of the electrolysis process, it is intended to develop a mathematical model that can simulate and assist in parameterization of the electrolysis performed by polymer membrane electrolytic cell. The experimental process to produce hydrogen via the cell membrane, aims to optimize the amount of gas produced using renewable energy with noncarbogenic causing no harm by producing gases deleterious to the environment. (author)

Flux Enhancement in Membrane Distillation Using Nanofiber Membranes

Directory of Open Access Journals (Sweden)

T. Jiříček

2016-01-01

Full Text Available Membrane distillation (MD is an emerging separation technology, whose largest application potential lies in the desalination of highly concentrated solutions, which are out of the scope of reverse osmosis. Despite many attractive features, this technology is still awaiting large industrial application. The main reason is the lack of commercially available membranes with fluxes comparable to reverse osmosis. MD is a thermal separation process driven by a partial vapour pressure difference. Flux, distillate purity, and thermal efficiency are always in conflict, all three being strictly connected with pore size, membrane hydrophobicity, and thickness. The world has not seen the ideal membrane yet, but nanofibers may offer a solution to these contradictory requirements. Membranes of electrospun PVDF were tested under various conditions on a direct contact (DCMD unit, in order to determine the optimum conditions for maximum flux. In addition, their performance was compared to commonly available PTFE, PE, and PES membranes. It was confirmed that thinner membranes have higher fluxes and a lower distillate purity and also higher energy losses via conduction across the membrane. As both mass and heat transfer are connected, it is best to develop new membranes with a target application in mind, for the specific membrane module and operational conditions.

Fabrication of electrospun nanofibrous membranes for membrane distillation application

KAUST Repository

Francis, Lijo

2013-02-01

Nanofibrous membranes of Matrimid have been successfully fabricated using an electrospinning technique under optimized conditions. Nanofibrous membranes are found to be highly hydrophobic with a high water contact angle of 130°. Field emission scanning electron microscopy and pore size distribution analysis revealed the big pore size structure of electrospun membranes to be greater than 2 μm and the pore size distribution is found to be narrow. Flat sheet Matrimid membranes were fabricated via casting followed by phase inversion. The morphology, pore size distribution, and water contact angle were measured and compared with the electrospun membranes. Both membranes fabricated by electrospinning and phase inversion techniques were tested in a direct contact membrane distillation process. Electrospun membranes showed high water vapor flux of 56 kg/m2-h, which is very high compared to the casted membrane as well as most of the fabricated and commercially available highly hydrophobic membranes. ©2013 Desalination Publications.

High Dehumidification Performance of Amorphous Cellulose Composite Membranes prepared from Trimethylsilyl Cellulose

KAUST Repository

Puspasari, Tiara

2018-04-11

Cellulose is widely regarded as an environmentally friendly, natural and low cost material which can significantly contribute the sustainable economic growth. In this study, cellulose composite membranes were prepared via regeneration of trimethylsilyl cellulose (TMSC), an easily synthesized cellulose derivative. The amorphous hydrophilic feature of the regenerated cellulose enabled fast permeation of water vapour. The pore-free cellulose layer thickness was adjustable by the initial TMSC concentration and acted as an efficient gas barrier. As a result, a 5,000 GPU water vapour transmission rate (WVTR) at the highest ideal selectivity of 1.1 x 106 was achieved by the membranes spin coated from a 7% (w/w) TMSC solution. The membranes maintained a 4,000 GPU WVTR with selectivity of 1.1 x 104 in the mixed-gas experiments, surpassing the performances of the previously reported composite membranes. This study provides a simple way to not only produce high performance membranes but also to advance cellulose as a low-cost and sustainable membrane material for dehumidification applications.

Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus

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Anaïs eCario

2015-10-01

Full Text Available The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80MPa. We analyzed the membrane lipids of T. barophilus by HPLC-MS as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol and, reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of non-polar lipids with an irregular polyisoprenoid carbon skeleton (polyunsaturated lycopane derivatives, suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other archaeon harboring a mixture of di- and tetraether lipids.

Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

Science.gov (United States)

Ho, J; Smith, S; Roh, H K

2014-01-01

A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.

Novel polyvinyl alcohol-bioglass 45S5 based composite nanofibrous membranes as bone scaffolds

International Nuclear Information System (INIS)

Shankhwar, Nisha; Kumar, Manishekhar; Mandal, Biman B.; Srinivasan, A.

2016-01-01

Composite nanofibrous membranes based on sol-gel derived 45SiO 2 24.5CaO 24.5 Na 2 O 6 P 2 O 5 (bioglass, BG) and 43SiO 2 24.5CaO 24.5 Na 2 O 6 P 2 O 5 2Fe 2 O 3 (magnetic bioglass, MBG) blended with polyvinyl alcohol (PVA) have been electrospun. These low cost membranes were mostly amorphous in structure with minor crystalline (sodium calcium phosphate) precipitates. All membranes were biodegradable. Among these, the composites exhibited higher tensile strength, better proliferation of human osteosarcoma MG63 cells and higher alkaline phosphatase enzyme activity than the bare PVA membrane, indicating their potential in bone tissue engineering. The magnetic PVA-MBG scaffold was also found to be a promising candidate for magnetic hyperthermia application. - Highlights: • Electrospun low-cost PVA-45S5 bioglass (BG) nanofibrous membranes • PVA-BG membranes containing 2 wt.% Fe 2 O 3 exhibit spontaneous magnetization. • BG fillers strongly enhanced mechanical strength and bioresponse of membranes. • Membranes show promise for bone scaffold and hyperthermia applications.

Membrane order in the plasma membrane and endocytic recycling compartment.

Science.gov (United States)

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Micropore structure stabilization in organosilica membranes by gaseous catalyst post-treatment

NARCIS (Netherlands)

Dral, A. Petra; van Eck, Ernst R.H.; Winnubst, Louis; ten Elshof, Johan E.

2018-01-01

A post-treatment involving repeated exposure to gaseous HCl alternated with heating is demonstrated to strongly accelerate the recently reported structural evolution in organically bridged silica networks. Films, powders and membranes derived from 1,2-bis(triethoxysilyl)ethane were exposed to

Membrane extraction with thermodynamically unstable diphosphonic acid derivatives

Science.gov (United States)

Horwitz, E.P.; Gatrone, R.C.; Nash, K.L.

1997-10-14

Thermodynamically-unstable complexing agents which are diphosphonic acids and diphosphonic acid derivatives (or sulphur containing analogs), like carboxyhydroxymethanediphosphonic acid and vinylidene-1,1-diphosphonic acid, are capable of complexing with metal ions, and especially metal ions in the II, III, IV, V and VI oxidation states, to form stable, water-soluble metal ion complexes in moderately alkaline to highly-acidic media. However, the complexing agents can be decomposed, under mild conditions, into non-organic compounds which, for many purposes are environmentally-nondamaging compounds thereby degrading the complex and releasing the metal ion for disposal or recovery. Uses for such complexing agents as well as methods for their manufacture are also described. 1 fig.

Uptake of DNA by cancer cells without a transfection reagent

Directory of Open Access Journals (Sweden)

Yanping Kong

Full Text Available Abstract Background Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. Methods A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer and THLE3 (normal liver cells after incubation overnight by counting radioactivity of the cells’ genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of “label it fluorescence in situ hybridization (FISH” from Mirus (USA. Results The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA’s size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. Conclusions In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA

The Role of Lipid Membranes in Life’s Origin

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David Deamer

2017-01-01

Full Text Available At some point in early evolution, life became cellular. Assuming that this step was required for the origin of life, there would necessarily be a pre-existing source of amphihilic compounds capable of assembling into membranous compartments. It is possible to make informed guesses about the properties of such compounds and the conditions most conducive to their self-assembly into boundary structures. The membranes were likely to incorporate mixtures of hydrocarbon derivatives between 10 and 20 carbons in length with carboxylate or hydroxyl head groups. Such compounds can be synthesized by chemical reactions and small amounts were almost certainly present in the prebiotic environment. Membrane assembly occurs most readily in low ionic strength solutions with minimal content of salt and divalent cations, which suggests that cellular life began in fresh water pools associated with volcanic islands rather than submarine hydrothermal vents.

cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response.

Directory of Open Access Journals (Sweden)

María Belén Mestre

Full Text Available Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla is the S. aureus-secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus-containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.

Inflammasome priming is similar for francisella species that differentially induce inflammasome activation.

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Mohammed G Ghonime

Full Text Available Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed Francisella strains (step 1, followed by ATP (step 2, we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica -LVS and F. tularensis Schu S4. This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

Invasion of Eukaryotic Cells by Legionella Pneumophila: A Common Strategy for all Hosts?

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Paul S Hoffman

1997-01-01

Full Text Available Legionella pneumophila is an environmental micro-organism capable of producing an acute lobar pneumonia, commonly referred to as Legionnaires’ disease, in susceptible humans. Legionellae are ubiquitous in aquatic environments, where they survive in biofilms or intracellularly in various protozoans. Susceptible humans become infected by breathing aerosols laden with the bacteria. The target cell for human infection is the alveolar macrophage, in which the bacteria abrogate phagolysosomal fusion. The remarkable ability of L pneumophila to infect a wide range of eukaryotic cells suggests a common strategy that exploits very fundamental cellular processes. The bacteria enter host cells via coiling phagocytosis and quickly subvert organelle trafficking events, leading to formation of a replicative phagosome in which the bacteria multiply. Vegetative growth continues for 8 to 10 h, after which the bacteria develop into a short, highly motile form called the ‘mature form’. The mature form exhibits a thickening of the cell wall, stains red with the Gimenez stain, and is between 10 and 100 times more infectious than agar-grown bacteria. Following host cell lysis, the released bacteria infect other host cells, in which the mature form differentiates into a Gimenez-negative vegetative form, and the cycle begins anew. Virulence of L pneumophila is considered to be multifactorial, and there is growing evidence for both stage specific and sequential gene expression. Thus, L pneumophila may be a good model system for dissecting events associated with the host-parasite interactions.

In vivo immunotoxicity evaluation of Gd2O3 nanoprobes prepared by laser ablation in liquid for MRI preclinical applications

Science.gov (United States)

Tian, Xiumei; Guan, Xiaoying; Luo, Ningqi; Yang, Fanwen; Chen, Dihu; Peng, Ye; Zhu, Jixiang; He, Fupo; Li, Li; Chen, Xiaoming

2014-09-01

Gd2O3 nanoprobes prepared by laser ablation in liquid can be used as magnetic resonance imaging contrast agent. However, their immunotoxicity in vivo remains unknown. In this article, the in vitro biocompatibility of the Gd2O3 nanoprobe was evaluated in terms of cell uptake, cell viability, and apoptosis. In vivo immunotoxicity was detected by monitoring the levels of the immunity mediator, cluster of differentiation (CD) markers in Balb/c mice. The results show that no in vitro cytotoxicity was observed, and no significant changes in the expression levels of CD206 and CD69 between the nanoprobe-injected group and the Gd-DTPA group in mice were observed. Importantly, the immunotoxicity data revealed significant differences in the expression levels of CD40, CD80, CD11b, and reactive oxygen species. In addition, transmission electron microscopy images showed that few Gd2O3 nanoprobes were localized in phagosomes by the endocytic pathway. In conclusion, the toxic effects of our Gd2O3 nanoprobe may be due to endocytosis during which the microstructure or ultrastructure of cells is slightly damaged and induces the generation of an oxidative stress reaction that further stimulates the innate immune response. Therefore, it is important to use a sensitive assay for the in vivo immunotoxicity measurements to evaluate the risk assessment of Gd2O3-based biomaterials at the molecular level.

Ionic liquids and their hosting by polymers for HT-PEMFC membranes

Energy Technology Data Exchange (ETDEWEB)

Hana, M.; Martinez, M.; Cointeaux, L.; Lepretre, J.C. [LEPMI-ELSA, PHELMA, UMR 5631, CNRS, Grenoble INP, UJF, Saint-Martin-d' Heres (France); Molmeret, Y.; El Kissi, N. [Laboratoire de Rheologie, UMR 5520 CNRS-INPG-UJF, ENSHMG, Grenoble (France); Teles, J.; Judeinstein, P. [Institut de Chimie Moleculaire et des Materiaux d' Orsay, CNRS 8182, Orsay (France); Iojoiu, C.; Sanchez, J.Y.

2010-10-15

The paper deals with proton-conducting ionic liquids (PCILs) for use, in combination with functional polymers, in membranes operating in high temperature PEMFC. Monoammoniums derived from monoamines and half-neutralised diamines were investigated in the form of triflates. Promising results were obtained with the half-neutralised diamine-based PCIL, its conduction being governed by both Grotthuss-like and vehicular mechanisms, the respective contributions of which depend on temperature. In addition, their blending with Nafion results in a distinct reinforcement of the membrane. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

The establishment of polarized membrane traffic in Xenopus laevis embryos.

Science.gov (United States)

Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

Giant plasma membrane vesicles: models for understanding membrane organization.

Science.gov (United States)

Levental, Kandice R; Levental, Ilya

2015-01-01

The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks.

Science.gov (United States)

Elliott, Michael H; Nash, Zack A; Takemori, Nobuaki; Fliesler, Steven J; McClellan, Mark E; Naash, Muna I

2008-01-01

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

Covalent Immobilization of Peroxidase onto Hybrid Membranes for the Construction of Optical Biosensor

Directory of Open Access Journals (Sweden)

Lyubov Yotova

2015-06-01

Full Text Available The aim of this study is to covalently immobilize horse radish peroxidase (HRP onto new hybrid membranes synthesized by the sol-gel method based on silica precursors, dendrimers and cellulose derivatives. This new system will be used for designing biosensor. For investigation of the properties of membranes, HRP was used as a modeling enzyme. Kinetic parameters, pH and temperature optimum were determined, and the structure of the membranes surface was examined. Results showed higher relative and residual activity of HRP immobilized onto membranes with cellulose acetate butyrate with high molecular weight CAB/H. This novel biosensor could offer a simple, cheap and rapid tool with enhanced sensing performance as well as having potentials to find application in medicine, pharmacy, food and process control and environmental monitoring.

Investigation of the Binding Site of CCR2 using 4-Azetidinyl-1-aryl-cyclohexane Derivatives: A Membrane Modeling and Molecular Dynamics Study

Energy Technology Data Exchange (ETDEWEB)

Kothandan, Gugan; Gadhe, Changdev G.; Cho, Seung Joo [Chosun Univ., Gwangju (Korea, Republic of)

2013-11-15

Chemokine receptor (CCR2) is a G protein-coupled receptor that contains seven transmembrane helices. Recent pharmaceutical research has focused on the antagonism of CCR2 and candidate drugs are currently undergoing clinical studies for the treatment of diseases like arthritis, multiple sclerosis, and type 2 diabetes. In this study, we analyzed the time dependent behavior of CCR2 docked with a potent 4-azetidinyl-1-aryl-cyclohexane (4AAC) derivative using molecular dynamics simulations (MDS) for 20 nanoseconds (ns). Homology modeling of CCR2 was performed and the 4AAC derivative was docked into this binding site. The docked model of selected conformations was then utilized to study the dynamic behavior of the 4AAC enzyme complexes inside lipid membrane. MDS of CCR2-16b of 4AAC complexes allowed us to refine the system since binding of an inhibitor to a receptor is a dynamic process and identify stable structures and better binding modes. Structure activity relationships (SAR) for 4AAC derivatives were investigated and reasons for the activities were determined. Probable binding pose for some CCR2 antagonists were determined from the perspectives of binding site. Initial modeling showed that Tyr49, Trp98, Ser101, Glu291, and additional residues are crucial for 4AAC binding, but MDS analysis showed that Ser101 may not be vital. 4AAC moved away from Ser101 and the hydrogen bonding between 4AAC and Ser101 vanished. The results of this study provide useful information regarding the structure-based drug design of CCR2 antagonists and additionally suggest key residues for further study by mutagenesis.

Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis.

Science.gov (United States)

Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

2013-09-06

Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy. Copyright © 2013 Elsevier Inc. All rights reserved.

Structure and physical properties of bio membranes and model membranes

International Nuclear Information System (INIS)

Tibor Hianik

2006-01-01

Bio membranes belong to the most important structures of the cell and the cell organelles. They play not only structural role of the barrier separating the external and internal part of the membrane but contain also various functional molecules, like receptors, ionic channels, carriers and enzymes. The cell membrane also preserves non-equilibrium state in a cell which is crucial for maintaining its excitability and other signaling functions. The growing interest to the bio membranes is also due to their unique physical properties. From physical point of view the bio membranes, that are composed of lipid bilayer into which are incorporated integral proteins and on their surface are anchored peripheral proteins and polysaccharides, represent liquid s crystal of smectic type. The bio membranes are characterized by anisotropy of structural and physical properties. The complex structure of bio membranes makes the study of their physical properties rather difficult. Therefore several model systems that mimic the structure of bio membranes were developed. Among them the lipid monolayers at an air-water interphase, bilayer lipid membranes, supported bilayer lipid membranes and liposomes are most known. This work is focused on the introduction into the physical word of the bio membranes and their models. After introduction to the membrane structure and the history of its establishment, the physical properties of the bio membranes and their models are stepwise presented. The most focus is on the properties of lipid monolayers, bilayer lipid membranes, supported bilayer lipid membranes and liposomes that were most detailed studied. This lecture has tutorial character that may be useful for undergraduate and graduate students in the area of biophysics, biochemistry, molecular biology and bioengineering, however it contains also original work of the author and his co-worker and PhD students, that may be useful also for specialists working in the field of bio membranes and model

Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues

OpenAIRE

1985-01-01

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room tem...

Protein profiles of hatchery egg shell membrane.

Science.gov (United States)

Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

2016-01-01

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

Directory of Open Access Journals (Sweden)

T. Jiříček

2017-01-01

Full Text Available Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest flux was achieved with the thinnest membranes and the best energy efficiency was achieved with the thickest membranes. All membranes had salt retention above 99%. Nanotechnology offers the potential to find modern solutions for desalination of waste waters, by introducing new materials with revolutionary properties, but new membranes must be developed according to the target application.

Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition