- A combined FTIR and DSC study on the bilayer-stabilising effect of electrostatic interactions in ion paired lipids. [Journal Article]
- CSColloids Surf B Biointerfaces 2018 May 17; 169:298-304
- Investigating lipid ion pair formation is important for understanding the mechanisms of lipid-mediated drug resistance in bacteria. In this study we have used the charged amphiphiles dipalmitoylphosp...
Investigating lipid ion pair formation is important for understanding the mechanisms of lipid-mediated drug resistance in bacteria. In this study we have used the charged amphiphiles dipalmitoylphosphatidylglycerol (DPPG) and dihexadecyldimethylammonium bromide (DHDAB), as a model to evaluate the formation of ion pairs by a combined Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analysis. FTIR was employed to study the environment of the DPPC headgroup phosphate and lipid/surfactant alkane chains, in vesicles formed by the two amphiphiles mixed in various molar ratios. An increase of the absorbance ratio of 1221-1201 cm-1 in the asymmetric phosphate stretching mode was found to follow a sigmoidal relationship with the proportion of DHDAB, increasing to a plateau above a DPPG/DHDAB 1:1 molar ratio of, providing evidence that the PG headgroup phosphate is involved in ion pairing. A consistent red shift was measured for the position of the symmetric CH2 stretch band for the lipid/surfactant 1:1 molar ratio mixture, which is indicative of an increased ordering of the hydrophobic chains. The DSC experiments yielded information about the thermotropic and the mixing behaviour of the lipid/surfactant systems. DPPG and DHDAB seem to form an ion pair with cluster compound characteristics at the equimolar ratio. Most interestingly, the DPPG/DHDAB 2:1 molar ratio mixture is characterized by strong intermolecular interactions, which result in a pronounced stabilization of the gel phase, possibly through the formation of a closely-associated ion triplet configuration in which the charges are delocalised across the headgroups.
- Co-mobility of GABARAP and Phosphatidylinositol 4-kinase 2A on cytoplasmic vesicles. [Journal Article]
- BBiochemistry 2018 May 24
- We previously reported that recruitment of the Type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important fo...
We previously reported that recruitment of the Type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome-lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under non-autophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A. C-terminal lipidation of GABARAP, which is essential for its autophagic activities, is not necessary for its recruitment to these PI4K2A-containing transport vesicles. However, a GABARAP truncation mutant lacking C-terminal residues 103-117 fails to bind to PI4K2A, is not recruited to cytoplasmic vesicles, and does not co-distribute with PI4K2A on subcellular organelles. These observations suggest that that the PI4K2A-GABARAP interaction plays a role in membrane trafficking both under autophagic and non-autophagic conditions.
- Manganese Transport and Toxicity in Polarized WIF-B Hepatocytes. [Journal Article]
- AJAm J Physiol Gastrointest Liver Physiol 2018 May 24
- Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to deter...
Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains while ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to > 250 M MnCl2. However, the hepatocytes were resistant to 4 h exposures of up to 500 M MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor brefeldin A did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile.
- p27Kip1 Modulates Axonal Transport by Regulating α-Tubulin Acetyltransferase 1 Stability. [Journal Article]
- CRCell Rep 2018 May 22; 23(8):2429-2442
- The protein p27Kip1 plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways ...
The protein p27Kip1 plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. Here, we show that p27Kip1 controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27Kip1 ortholog, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27Kip1 stabilizes the α-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport.
- Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4 Translocation and Performance in Skeletal Muscles. [Journal Article]
- CRCell Rep 2018 May 22; 23(8):2354-2364
- Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanis...
Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanistic interactions between working skeletal muscles and locally recruited neutrophils expressing IL-1β, which supports muscle performance through priming exercise-dependent GLUT4 translocation. Thus, during exercise, both IL-1α/β-deficient and neutrophil-depleted mice similarly exhibit increased fatigability associated with impaired muscle glucose homeostasis due to GLUT4 dysregulation. Deficiency of IL-1-producing neutrophils results in intrinsic abnormalities represented by aberrant Rac1 signaling and irregular GLUT4-storage vesicles, suggesting that these properties are maintained by local IL-1 produced by recruited neutrophils upon exercise, possibly on a daily basis. We propose that neutrophils are highly engaged in skeletal muscle performance via IL-1 regulation, which coordinates favorable inflammatory microenvironments supporting muscle glucose metabolism.
- Uncovering cryptic diversity of Lyngbya: the new tropical marine cyanobacterial genus Dapis (Oscillatoriales). [Journal Article]
- JPJ Phycol 2018 May 23
- Cyanobacteria comprise an extraordinarily diverse group of microorganisms and, as revealed by increasing molecular information, this biodiversity is even more extensive than previously estimated. In ...
Cyanobacteria comprise an extraordinarily diverse group of microorganisms and, as revealed by increasing molecular information, this biodiversity is even more extensive than previously estimated. In this sense, the cyanobacterial genus Lyngbya is a highly polyphyletic group composed of many unrelated taxa with morphological similarities. In this study, the new genus Dapis was erected from the genus Lyngbya, based on a combined molecular, chemical, and morphological approach. Herein, two new species of cyanobacteria are described: D. pleousa and D. pnigousa. Our analyses found these species to be widely distributed and abundant in tropical and subtropical marine habitats. Seasonally, both species have the ability to form extensive algal blooms in marine habitats: D. pleousa in shallow-water, soft bottom habitats and D. pnigousa on coral reefs below depths of 10 m. Electron microscopy showed that D. pleousa contains gas vesicles, a character not previously reported in Lyngbya. These gas vesicles, in conjunction with a mesh-like network of filaments that trap oxygen released from photosynthesis, provides this species with an unusual mechanism to disperse in coastal marine waters, allowing D. pleousa to be present in both benthic and planktonic forms. In addition, both D. pleousa and D. pnigousa contained nitrogen-fixing genes as well as bioactive secondary metabolites. Several specimens of D. pnigousa biosynthesized the secondary metabolite lyngbic acid, a molecule that has also been isolated from many other marine cyanobacteria. Dapis pleousa consistently produced the secondary metabolite malyngolide, which may provide a promising chemotaxonomic marker for this species. This article is protected by copyright. All rights reserved.
- Cloning and expression of nlpA gene as DNA vaccine candidate against Acinetobacter baumannii. [Journal Article]
- MBMol Biol Rep 2018 May 22
- Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria that cause infections with high rate of death. This bacterium is one the common causes of infection worldwide leading to end...
Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria that cause infections with high rate of death. This bacterium is one the common causes of infection worldwide leading to endemic and epidemic nosocomial infections. Despite many efforts, there is no effective vaccine against A. baumannii. As NlpA is one of the important antigenic factors in biogenesis of outer membrane vesicles, and OMV-based reported vaccines in A. baumannii stimulated the immune responses, this study was aimed to clone and express nlpA gene in eukaryotic HDF cells and evaluate the induced immunization following the administration of resulting construct as DNA vaccine in BALB/c mice. The nlpA gene of A. baumannii was amplified using PCR. The PCR product was then cloned and subcloned into the pTZ57R/T and pEGFP-C2 vectors respectively. The cloning was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The pEGFP-C2-nlpA recombinant plasmid was transferred into the HDF cells using electroporation and the expression of target gene was validated by RT-PCR. The recombinant construct was injected to BALB/c mice through three IM injections and the levels of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 were determined using ELISA assay. The A. baumannii nlpA gene was amplified during PCR as 867 bp band which was successfully cloned in pEGFP-C2-nlpA vector. Obtained data from RT-PCR and presence of the 867 bp fragment in transformed HDF cells confirmed the nlpA gene expression. Following the injection of pEGFP-C2-nlpA showed the increased level of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 in serum of immunized mice. Overall, through this study recombinant pEGFP-C2-nlpA was generated and successfully expressed the A. baumannii nlpA gene in eukaryotic cells. Additionally, our in vivo study confirmed that the recombinant construct capable to induce the immune response in immunized mice. These findings suggest the pEGFP-C2-nlpA may be considered as DNA vaccine candidate against A. baumannii.
- Intricate relationships between naked viruses and extracellular vesicles in the crosstalk between pathogen and host. [Review]
- SISemin Immunopathol 2018 May 22
- It is a long-standing paradigm in the field of virology that naked viruses cause lysis of infected cells to release progeny virus. However, recent data indicate that naked virus types of the Picornav...
It is a long-standing paradigm in the field of virology that naked viruses cause lysis of infected cells to release progeny virus. However, recent data indicate that naked virus types of the Picornaviridae and Hepeviridae families can also leave cells via an alternative route involving enclosure in fully host-derived lipid bilayers. The resulting particles resemble extracellular vesicles (EV), which are 50 nm-1 μm vesicles released by all cells. These EV contain lipids, proteins, and RNA, and generally serve as vehicles for intercellular communication in various (patho)physiological processes. EV can act as carriers of naked viruses and as invisibility cloaks to evade immune attacks. However, the exact combination of virions and host-derived molecules determines how these virus-containing EV affect spread of infection and/or triggering of antiviral immune responses. An underexposed aspect in this research area is that infected cells likely release multiple types of virus-induced and constitutively released EV with unique molecular composition and function. In this review, we identify virus-, cell-, and environment-specific factors that shape the EV population released by naked virus-infected cells. In addition, current findings on the formation and molecular composition of EV induced by different virus types will be compared and placed in the context of the widely proven heterogeneity of EV populations and biases caused by different EV isolation methodologies. Close interactions between the fields of EV biology and virology will help to further delineate the intricate relationship between EV and naked viruses and its relevance for viral life cycles and outcomes of viral infections.
- Existing and novel biomarkers for precision medicine in systemic sclerosis. [Review]
- NRNat Rev Rheumatol 2018 May 22
- The discovery and validation of biomarkers resulting from technological advances in the analysis of genomic, transcriptomic, lipidomic and metabolomic pathways involved in the pathogenesis of complex...
The discovery and validation of biomarkers resulting from technological advances in the analysis of genomic, transcriptomic, lipidomic and metabolomic pathways involved in the pathogenesis of complex human diseases have led to the development of personalized and rationally designed approaches for the clinical management of such disorders. Although some of these approaches have been applied to systemic sclerosis (SSc), an unmet need remains for validated, non-invasive biomarkers to aid in the diagnosis of SSc, as well as in the assessment of disease progression and response to therapeutic interventions. Advances in global transcriptomic technology over the past 15 years have enabled the assessment of microRNAs that circulate in the blood of patients and the analysis of the macromolecular content of a diverse group of lipid bilayer membrane-enclosed extracellular vesicles, such as exosomes and other microvesicles, which are released by all cells into the extracellular space and circulation. Such advances have provided new opportunities for the discovery of biomarkers in SSc that could potentially be used to improve the design and evaluation of clinical trials and that will undoubtedly enable the development of personalized and individualized medicine for patients with SSc.
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- Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation. [Journal Article]
- NCNat Commun 2018 May 22; 9(1):2013
- The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT m...
The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.