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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2012

Community Structure and Functional Gene Profile of Bacteria on Healthy and Diseased Thalli of the Red Seaweed Delisea pulchra

Disease is increasingly viewed as a major factor in the ecology of marine communities and its impact appears to be increasing with environmental change, such as global warming. The temperate macroalga Delisea pulchra bleaches in Southeast Australia during warm summer periods, a phenomenon which previous studies have indicated is caused by a temperature induced bacterial disease. In order to better understand the ecology of this disease, the bacterial communities associated with threes type of samples was investigated using 16S rRNA gene and environmental shotgun sequencing: 1) unbleached (healthy) D. pulchra 2) bleached parts of D. pulchra and 3) apparently healthy tissue adjacent to bleached regions. Phylogenetic differences between healthy and bleached communities mainly reflected relative changes in the taxa Colwelliaceae, Rhodobacteraceae, Thalassomonas and Parvularcula. Comparative metagenomics showed clear difference in the communities of healthy and diseased D. pulchra as reflected by changes in functions associated with transcriptional regulation, cation/multidrug efflux and non-ribosomal peptide synthesis. Importantly, the phylogenetic and functional composition of apparently healthy tissue adjacent to bleached sections of the thalli indicated that changes in the microbial communities already occur in the absence of visible tissue damage. This shift in unbleached sections might be due to the decrease in furanones, algal metabolites which are antagonists of bacterial quorum sensing. This study reveals the complex shift in the community composition associated with bleaching of Delisea pulchra and together with previous studies is consistent with a model in which elevated temperatures reduce levels of chemical defenses in stressed thalli, leading to colonization or proliferation by opportunistic pathogens or scavengers.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2013

Bacterial Diversity in Two Neonatal Intensive Care Units (NICUs)

Infants in Neonatal Intensive Care Units (NICUs) are particularly susceptible to opportunistic infection. Infected infants have high mortality rates, and survivors often suffer life-long neurological disorders. The causes of many NICU infections go undiagnosed, and there is debate as to the importance of inanimate hospital environments (IHEs) in the spread of infections. Here, we used culture-independent next-generation sequencing to survey bacterial diversity in two San Diego NICUs and to track the sources of microbes in these environments. Thirty IHE samples were collected from two Level-Three NICU facilities. We extracted DNA from these samples and amplified the bacterial small subunit (16S) ribosomal RNA gene sequence using ‘universal’ barcoded primers. The purified PCR products were pooled into a single reaction for pyrosequencing, and the data were analyzed using QIIME. On average, we detected 93+/-39 (mean +/- standard deviation) bacterial genera per sample in NICU IHEs. Many of the bacterial genera included known opportunistic pathogens, and many were skin-associated (e.g., Propionibacterium). In one NICU, we also detected fecal coliform bacteria (Enterobacteriales) in a high proportion of the surface samples. Comparison of these NICU-derived sequences to previously published high-throughput 16S rRNA amplicon studies of other indoor environments (offices, restrooms and healthcare facilities), as well as human- and soil-associated environments, found the majority of the NICU samples to be similar to typical building surface and air samples, with the notable exception of the IHEs which were dominated by Enterobacteriaceae. Our findings provide evidence that NICU IHEs harbor a high diversity of human-associated bacteria and demonstrate the potential utility of molecular methods for identifying and tracking bacterial diversity in NICUs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Visualizing and analyzing 3D biomolecular structures using Mol* at RCSB.org: Influenza A H5N1 virus proteome case study

The easiest and often most useful way to work with experimentally determined or computationally predicted structures of biomolecules is by viewing their three-dimensional (3D) shapes using a molecular visualization tool. Mol* was collaboratively developed by RCSB Protein Data Bank (RCSB PDB, RCSB.org) and Protein Data Bank in Europe (PDBe, PDBe.org) as an open-source, web-based, 3D visualization software suite for examination and analyses of biostructures. It is capable of displaying atomic coordinates and related experimental data of biomolecular structures together with a variety of annotations, facilitating basic and applied research, training, education, and information dissemination. Across RCSB.org, the RCSB PDB research-focused web portal, Mol* has been implemented to support single-mouse-click atomic-level visualization of biomolecules (e.g., proteins, nucleic acids, carbohydrates) with bound cofactors, small-molecule ligands, ions, water molecules, or other macromolecules. RCSB.org Mol* can seamlessly display 3D structures from various sources, allowing structure interrogation, superimposition, and comparison. Using influenza A H5N1 virus as a topical case study of an important pathogen, we exemplify how Mol* has been embedded within various RCSB.org tools—allowing users to view polymer sequence and structure-based annotations integrated from trusted bioinformatics data resources, assess patterns and trends in groups of structures, and view structures of any size and compositional complexity. In addition to being linked to every experimentally determined biostructure and Computed Structure Model made available at RCSB.org, Standalone Mol* is freely available for visualizing any atomic-level or multi-scale biostructure at rcsb.org/3d-view.

3D biostructure↗
DOE OSTI2012

Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

08 HYDROGEN↗
DOE OSTI2015

Stent-Graft Placement with Early Debridement and Antibiotic Treatment for Femoral Pseudoaneurysms in Intravenous Drug Addicts

PurposeExplore the application of endovascular covered stent-graft (SG) placement in femoral pseudoaneurysms in intravenous drug addicts.Materials and MethodsWe evaluated a consecutive series of pseudoaneurysm in intravenous drug addicts treated with SGs from August 2010 to December 2013.Results15 patients with 16 arterial pseudoaneurysms were enrolled in this study. All were males with a mean age of 36.9 years. Hemorrhage was the most common reason (93.8 %) for seeking medical care, and 3 of these patients were in hemorrhagic shock at admission. All patients received broad-spectrum antibiotics, and debridement and drainage were implemented after SG placement. 7 of the 13 cases which had microbiologic results showed mixed infections, while gram-negative bacteria were the major pathogens. Except for 2 patients, who were lost to follow-up, two new pseudoaneurysms formed due to delayed debridement, and one stent thrombosis occurred, none of the remaining cases had SG infection or developed claudication.ConclusionsSG placement controls massive hemorrhage rapidly, gives enough time for subsequent treatment for pseudoaneurysms due to intravenous drug abuse, and reduces the incidence of postoperative claudication. With appropriate broad-spectrum antibiotics and early debridement, the incidence of SG infection is relatively low. It is an effective alternative especially as temporary bridge measure for critical patients. However, the high cost, uncertain long-term prospects, high demand for medical adherence, and the risk of using the conduits for re-puncture call for a cautious selection of patients. More evidence is required for the application of this treatment.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2014

Intronic and plasmid-derived regions contribute to the large mitochondrial genome sizes of Agaricomycetes

Sizes of mitochondrial genomes vary extensively between fungal species although they typically contain a conserved set of core genes. We have characterised the mitochondrial genome of the conifer root rot pathogen Heterobasidion irregulare and compared the size, gene content and structure of 20 Basidiomycete mitochondrial genomes. The mitochondrial genome of H. irregulare was 114, 193 bp and contained a core set of 15 protein coding genes, two rRNA genes and 26 tRNA genes. In addition, we found six non-conserved open reading frames (ORFs) and four putative plasmid genes clustered in three separate regions together with 24 introns and 14 intronic homing endonuclease genes, unequally spread across seven of the core genes. The size differences among the 20 Basidiomycetes can largely be explained by length variation of intergenic regions and introns. The Agaricomycetes contained the nine largest mitochondrial genomes in the Basidiomycete group and Agaricomycete genomes are significantly (p < 0.001) larger than the other Basidiomycetes. A feature of the Agaricomycete mitochondrial genomes in this study was the simultaneous occurrence of putative plasmid genes and non-conserved ORFs, with Cantharellus cibarius as only exception, where no non-conserved ORF was identified. This indicates a mitochondrial plasmid origin of the non-conserved ORFs or increased mitochondrial genome dynamics of species harbouring mitochondrial plasmids. We hypothesise that two independent factors are the driving forces for large mitochondrial genomes: the homing endonuclease genes in introns and integration of plasmid DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bayesian time-varying occupancy model for West Nile virus in Ontario, Canada

Occupancy models determine the true presence or absence of a species by adjusting for imperfect detection in surveys. They often assume that species presences can be detected only if sites are occupied during a sampling season. We extended these models to estimate occupancy rates that vary throughout a sampling season as well as account for spatial dependence among sites. For these methods, we constructed a fast Gibbs sampler with the Pólya-Gamma augmentation strategy to conduct inference on covariate effects. We applied these methods to evaluate how environmental conditions and surveillance practices are associated with the presence of West Nile virus in mosquito traps across Ontario, Canada from 2002 to 2017. We found that urban land cover and warm temperatures drove viral occupancy, whereas viral testing on pools with higher proportions of Culex mosquitoes was more likely to result in a positive test for West Nile virus. Models with time-varying occupancy effects achieved much lower Watanabe-Akaike information criteria than models without such effects. Our final model had strong predictive performance on test data that included some of the most extreme seasons, demonstrating the promise of these methods in the study of pathogens spread by mosquito vectors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Preparation and Characterization of Polyaniline and Ag/ Polyaniline Composite Nanoporous Particles and Their Antimicrobial Activities

Polyaniline (PANI) and Ag/PANI nanoporous composite were prepared by an oxidative polymerization method. The oxidation process of PANI nanoparticles was occurred using (NH{sub 4}){sub 2}S{sub 2}O{sub 8} while the oxidation process of Ag/PANI nanoporous composite was occurred using AgNO{sub 3} under the effect of artificial radiation. The structural, morphological, and optical properties of the PANI and Ag/PANI nanoporous structures were studied using different characterization tools. The results confirm the formation of polycrystalline nanoporous PANI and spherical nanoporous composite of Ag/PANI particles. Antibacterial activity tests against gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, and gram-negative bacteria, Escherichia coli, and Salmonella species were carried out using different concentrations of PANI nanoparticles and Ag/PANI nanoporous composites. PANI has not antibacterial effect against all studied pathogens. In contrast, Ag/PANI nanoporous composites possessed antibacterial activity that is identified by the zone of inhibition. The inhibition zones of bacteria are in order; Salmonella species > S. aureus > B. subtilis > E. coli. The inhibition zones of all bacteria increased with increasing concentrations of Ag/PANI nanoporous composites from 200 to 400 ppm then decreased with further increasing of the dose concentrations to 600 ppm. Finally, a simplified mechanism based on the electrostatic attraction is presented to describe the antimicrobial activity of Ag/PANI nanoporous composite.

36 MATERIALS SCIENCE↗
DOE OSTI2019

Synthesis of Melanin Mediated Silver Nanoparticles from Aeromonas sp. SNS Using Response Surface Methodology: Characterization with the Biomedical Applications and Photocatalytic Degradation of Brilliant Green

Melanin is ubiquitous in nature and has wide applications in cosmetics, agriculture, and medicine. The synthesized melanin from bacterium Aeromonas sp. SNS was further used as capping and reducing agents for synthesis of silver nanoparticles (AgNPs). The influence of the experimental parameters (AgNO{sub 3}, melanin concentrations, and temperature) and their interactions on the nanoparticle synthesis was optimized using response surface methodology (RSM). The Central Composite Design (CCD) with three independent variables was optimized for the effective synthesis of AgNPs. The optimized synthesis of AgNPs was achieved at the shortest time of 14.12 h in the presence of 2.62 mM AgNO{sub 3}, and 32.30 µg ml{sup −1} melanin concentration at 54.86 °C temperature. The synthesized AgNPs were characterized by means of UV–visible spectroscopy, FTIR, SEM, TEM, and PSD respectively. The AgNPs exhibited excellent antimicrobial activity against human and food-related pathogens. These AgNPs also have strong antioxidant potential which was estimated by DPPH, DMPD and FRAP radical scavenging assays. The 92.62% photocatalytic degradation of 250 PPM brilliant green was observed in 120 min. The present finding accelerates the melanin associated AgNPs could be used in the cosmetic and pharmaceutical industries as well as in textile industries as they have superior antimicrobial, antioxidant, and photocatalytic activity.

36 MATERIALS SCIENCE↗
DOE OSTI2011

Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein

Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone 1HN, 15N, 13CO, and 13Ca, as well as side chain 13Cb, methyl (Ile-d1, Leu, Val), amide (Asn, Gln), and indole NH (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Hemagglutinating virus of Japan envelope (HVJ-E) can enhance the immune responses of swine immunized with killed PRRSV vaccine

Highlights: Black-Right-Pointing-Pointer We investigated the immunoadjuvant effects of HVJ-E on killed PRRSV vaccine. Black-Right-Pointing-Pointer HVJ-E enhanced the humoral and cellular responses of the piglets to PRRSV. Black-Right-Pointing-Pointer It is suggested that HVJ-E could be developed as a new-type adjuvant for mammals. -- Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically detrimental pig pathogen that causes significant losses for the pig industry. The immunostimulatory effects of hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy and the adjuvant efficacy of HVJ-E have been previously evaluated. The objective of this study was to investigate the adjuvant effects of HVJ-E on immunization with killed PRRSV vaccine, and to evaluate the protective effects of this immunization strategy against virulent PRRSV infection in piglets. Next, the PRRSV-specific antibody response, lymphocyte proliferation, PRRSV-specific IL-2, IL-10 and IFN-{gamma} production, and the overall protection efficacy were evaluated to assess the immune responses of the piglets. The results showed that the piglets inoculated simultaneously with killed PRRSV vaccine and HVJ-E had a significantly stronger immune response than those inoculated with killed PRRSV vaccine alone. Our results suggest that HVJ-E could be employed as an effective adjuvant to enhance the humoral and cellular responses of piglets to PRRSV.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Highlights: Black-Right-Pointing-Pointer Mycoplasma mobile was observed in buffer with the Atmospheric Scanning Electron Microscope. Black-Right-Pointing-Pointer Characteristic protein localizations were visualized using immuno-labeling. Black-Right-Pointing-Pointer M. mobile attached to sialic acid on the SiN film surface within minutes. Black-Right-Pointing-Pointer Cells were observed at low concentrations. Black-Right-Pointing-Pointer ASEM should promote study and early-stage diagnosis of mycoplasma. -- Abstract: Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3 {mu}m-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated by hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt contributes to hypoxic stress-induced TLR4 expression at least partly through the regulation of HIF-1 activation. These reveal a novel mechanism for regulation of TLR4 expression upon hypoxic stress and provide a therapeutic target for chronic diseases related to hypoxic stress.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy

Highlights: Black-Right-Pointing-Pointer New kyotorphin derivatives have antimicrobial properties against S. aureus. Black-Right-Pointing-Pointer Atomic force microscopy show membrane disturbing effects of KTP-NH{sub 2} and IbKTP-NH{sub 2}. Black-Right-Pointing-Pointer None of the KTP derivatives are hemolytic. Black-Right-Pointing-Pointer The minimal peptidic sequence with antimicrobial activity is Tyr-Arg, if amidated. -- Abstract: Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH{sub 2}, IbKTP, IbKTP-NH{sub 2} - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

Highlights: Black-Right-Pointing-Pointer We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. Black-Right-Pointing-Pointer The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. Black-Right-Pointing-Pointer The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. Black-Right-Pointing-Pointer The OgUBC1 could protect disruption of plant cells by UV-B radiation. Black-Right-Pointing-Pointer OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells infected with M.tuberculosis and may represent a molecular target for promoting cell survival during infection by respiratory pathogens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Electron microscopic visualization of autophagosomes induced by infection of human papillomavirus pseudovirions

Highlights: •HPV16 pseudovirions (16PsVs) infection induces an autophagy response. •The autophagy was analyzed by transmission electron microscope (TEM). •TEM showed the double-membrane vesicles in HeLa cells inoculated with 16PsVs. •These vesicles incorporated 16PsVs particles in the lumen. •These results imply that autophagosomes are generated from the plasma membrane. -- Abstract: Autophagy is a bulk degradation process for subcellular proteins and organelles to manage cell starvation. Autophagy is associated with the formation of autophagosomes and further functions as a defense mechanism against infection by various pathogens. Human papillomavirus (HPV) infection induces an autophagy response, such as up-regulation of marker proteins for autophagy, in host keratinocytes. However, direct microscopic evidence for autophagy induction by HPV infection is still lacking. Here, I report an electron microscopic analysis of autophagosomes elicited by the entry of HPV pseudovirions (PsVs). HeLa cells showed enhanced infectivity for PsVs of HPV type 16 (16PsVs) when treated with an autophagy inhibitor, suggesting the involvement of autophagy in HPV infection. In HeLa cells inoculated with 16PsVs, transmission electron microscopy showed the presence of cup-shaped, double-membrane vesicles (phagophores) and double-membrane-bound vesicles, which are typical structures of autophagosomes. These double-membrane vesicles displayed a large lumen volume and incorporated 10–50 16PsVs particles in the lumen. These results demonstrate that autophagy is indeed induced during the HPV16 entry process and imply that autophagosomes are generated from the plasma membrane by HPV infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

60 APPLIED LIFE SCIENCES↗