Nanopods: A New Bacterial Structure and Mechanism for Deployment of Outer Membrane Vesicles

Background: Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;). Methodology/Principal Findings: We have identified a new bacterial surface structure, termed a "nanopod", that is a conduit for projecting OMV significant distances (e.g., ≥6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods. Conclusions/Significance: Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms

The FIREBIRD Instrument for Relativistic Electrons: Enabling Technologies for a Fast High-Sensitivity, Low-Power Space Weather Radiation Payload

Miniaturized instrument payloads on small satellite and nanosatellite platforms that are deployed in low Earth orbit are demonstrating cost effective weather monitoring platforms with increased temporal and spatial resolution compared to larger weather satellites. The NASA Earth Decadal Survey [1] states that improving the revisit time of microwave radiometers would significantly improve weather forecasting. Radiometers such as the Advanced Technology Microwave Sounder (ATMS) on Suomi National Polar-orbiting Partnership (Suomi-NPP) and the Joint Polar Satellite System-1 (JPSS-1), now NOAA-20, provide an average revisit rate of 7.6 hours; however, a constellation of six CubeSats in three orbital Low Earth Orbit (LEO) planes with microwave radiometers such as the Time-Resolved Observations of Precipitations structure and storm Intensity with a Constellation of Smallsats (TROPICS) mission would provide a refresh rate of better than 60 minutes. In order to effectively use CubeSats in a constellation as a weather monitoring platform, calibration must be used to provide measurements consistent with state of the art measurements, such as ATMS that has a NeDT at 300K of 0.5-3.0K [2]. In this work, we use the Joint Center for Satellite Data Assimilation (JCSDA) Community Radiative Transfer Model (CRTM) to simulate brightness temperatures (https://www.jcsda.noaa.gov/projects_crtm.php), which are used to assess miniaturized microwave radiometer radiometric biases. CRTM is a fast radiative transfer model that uses Fortran functions, structure variables, and coefficient data of the modeled sensor to simulate radiances. The user inputs surface characteristics, scan angles, and atmospheric profiles from sources such as radiosondes, Numerical Weather Prediction (NWP) models, and Global Positioning System Radio Occultation (GPSRO) measurements. The output of CRTM is a simulated brightness temperature that is used to correct radiometric biases in order to meet required instrument NeDT performance. We use radiosonde, GPSRO, and NWP ERA-5 atmospheric profiles in CRTM and compare the results to ATMS brightness temperatures and find an average difference in brightness temperature of 1.95 K, which is comparable to ATMS Integrated Calibration/Validation System (https://www.star.nesdis.noaa.gov/icvs/status_NPP_ATMS.php) reports which show channel bias variations of up to 2 K. We take a similar approach to provide calibration for the Micro-sized Microwave Atmospheric Satellite-2A (MicroMAS-2A), a 3U CubeSat that was launched on January 11th, 2018. MicroMAS-2A carries a 1U 10-channel passive microwave radiometer that provides imagery near 90 and 206 GHz, temperature sounding near 118 GHz, and moisture sounding near 183 GHz. We develop an approach for comparing MicroMas-2A brightness temperatures to radiosonde, GPSRO, and NWP ERA5 atmospheric profiles. Due to the scarcity of GPSRO and radiosonde profiles near the MicroMAS-2A data segments, we determine that NWP models will be the best option for radiance validation. After the next stage of calibration of MicroMAS-2A is completed, we will compare CRTM simulated radiances from ERA profiles to the initial sensor data, with expected results of channel bias variations of \u3c 2 K

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses

Purpose: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. Methods: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. Results: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. Conclusions: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles

Pregnancy rate and reproductive hormones in humpback whale blubber: Dominant form of progesterone differs during pregnancy

To better understand reproductive physiology of humpback whales Megaptera novaeangliae that reside in Hawai’i and Alaska, enzyme immunoassays were validated for both progesterone and testosterone in free-ranging and stranded animals (n = 185 biopsies). Concentrations were analyzed between different depths of large segments of blubber taken from skin to muscle layers of stranded female (n = 2, 1 pregnant, 1 non-pregnant) and male (n = 1) whales. Additionally, progesterone metabolites were identified between pregnant (n = 1) and non-pregnant (n = 3) females using high pressure liquid chromatography (HPLC). Progesterone concentrations were compared between juvenile (i.e., sexually immature), lactating, and pregnant females, and male whales, and pregnancy rates of sexually mature females were calculated. Based on replicate samples from ship struck animals collected at 7 depth locations, blubber containing the highest concentration of progesterone was located 1 cm below the skin for females, and the highest concentration of testosterone was in the skin layer of one male whale. HPLC of blubber samples of pregnant and non-pregnant females contain different immunoreactive progesterone metabolites, with the non-pregnant female eluate comprised of a more polar, and possibly conjugated, form of progesterone than the pregnant female. In females, concentrations of progesterone were highest in the blubber of pregnant (n = 28, 28.6 ± 6.9 ng/g), followed by lactating (n = 16, 0.9 ± 0.1 ng/g), and female juvenile (n = 5, 1.0 ± 0.2 ng/g) whales. Progesterone concentrations in male (n = 24, 0.6 ng/g ± 0.1 ng/g) tissues were the lowest all groups, and not different from lactating or juvenile females. Estimated summer season pregnancy rate among sexually mature females from the Hawai’i stock of humpback whales was 0.562 (95 % confidence interval 0.528–0.605). For lactating females, the year-round pregnancy rate was 0.243 (0.09–0.59), and varies depending on the threshold of progesterone assumed for pregnancy in the range between 3.1 and 28.5 ng/g. Our results demonstrate the synergistic value added when combining immunoreactive assays, HPLC, and long-term sighting histories to further knowledge of humpback whale reproductive physiology.United States Office of Naval Research. National Marine Fisheries. Biomedical Learning and Student Training (BLaST) Program through the National Institute of General Medical Sciences of the National Institutes of Health. Idea Network for Biomedical Research and Education (INBRE).Abstract -- 1. Introduction -- 2. Methods -- 3. Results -- 4. Discussion -- Acknowledgements -- References -- Tables & Figures.Ye

The Interface Region Imaging Spectrograph (IRIS)

The Interface Region Imaging Spectrograph (IRIS) small explorer spacecraft provides simultaneous spectra and images of the photosphere, chromosphere, transition region, and corona with 0.33-0.4 arcsec spatial resolution, 2 s temporal resolution and 1 km/s velocity resolution over a field-of-view of up to 175 arcsec x 175 arcsec. IRIS was launched into a Sun-synchronous orbit on 27 June 2013 using a Pegasus-XL rocket and consists of a 19-cm UV telescope that feeds a slit-based dual-bandpass imaging spectrograph. IRIS obtains spectra in passbands from 1332-1358, 1389-1407 and 2783-2834 Angstrom including bright spectral lines formed in the chromosphere (Mg II h 2803 Angstrom and Mg II k 2796 Angstrom) and transition region (C II 1334/1335 Angstrom and Si IV 1394/1403 Angstrom). Slit-jaw images in four different passbands (C II 1330, Si IV 1400, Mg II k 2796 and Mg II wing 2830 Angstrom) can be taken simultaneously with spectral rasters that sample regions up to 130 arcsec x 175 arcsec at a variety of spatial samplings (from 0.33 arcsec and up). IRIS is sensitive to emission from plasma at temperatures between 5000 K and 10 MK and will advance our understanding of the flow of mass and energy through an interface region, formed by the chromosphere and transition region, between the photosphere and corona. This highly structured and dynamic region not only acts as the conduit of all mass and energy feeding into the corona and solar wind, it also requires an order of magnitude more energy to heat than the corona and solar wind combined. The IRIS investigation includes a strong numerical modeling component based on advanced radiative-MHD codes to facilitate interpretation of observations of this complex region. Approximately eight Gbytes of data (after compression) are acquired by IRIS each day and made available for unrestricted use within a few days of the observation.Comment: 53 pages, 15 figure

Cystinosin, MPDU1, SWEETs and KDELR Belong to a Well-Defined Protein Family with Putative Function of Cargo Receptors Involved in Vesicle Trafficking

Classification of proteins into families based on remote homology often helps prediction of their biological function. Here we describe prediction of protein cargo receptors involved in vesicle formation and protein trafficking. Hidden Markov model profile-to-profile searches in protein databases using endoplasmic reticulum lumen protein retaining receptors (KDEL, Erd2) as query reveal a large and diverse family of proteins with seven transmembrane helices and common topology and, most likely, similar function. Their coding genes exist in all eukaryota and in several prokaryota. Some are responsible for metabolic diseases (cystinosis, congenital disorder of glycosylation), others are candidate genes for genetic disorders (cleft lip and palate, certain forms of cancer) or solute uptake and efflux (SWEETs) and many have not yet been assigned a function. Comparison with the properties of KDEL receptors suggests that the family members could be involved in protein trafficking and serve as cargo receptors. This prediction sheds new light on a range of biologically, medically and agronomically important proteins and could open the way to discovering the function of many genes not yet annotated. Experimental testing is suggested

Evolution of metabolic divergence in <i>Pseudomonas aeruginosa</i> during long-term infection facilitates a proto-cooperative interspecies interaction

The effect of polymicrobial interactions on pathogen physiology and how it can act either to limit pathogen colonization or to potentiate pathogen expansion and virulence are not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found together in polymicrobial human infections. However, we have previously shown that the interactions between these two bacterial species are strain dependent. Whereas P. aeruginosa PAO1, a commonly used laboratory strain, effectively suppressed S. aureus growth, we observed a commensal-like interaction between the human host-adapted strain, DK2-P2M24-2003, and S. aureus. In this study, characterization by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) and mass spectral (MS) molecular networking revealed a significant metabolic divergence between P. aeruginosa PAO1 and DK2-P2M24-2003, which comprised several virulence factors and signaling 4-hydroxy-2-alkylquinoline (HAQ) molecules. Strikingly, a further modulation of the HAQ profile was observed in DK2-P2M24-2003 during interaction with S. aureus, resulting in an area with thickened colony morphology at the P. aeruginosa–S. aureus interface. In addition, we found an HAQ-mediated protection of S. aureus by DK2-P2M24-2003 from the killing effect of tobramycin. Our findings suggest a model where the metabolic divergence manifested in human host-adapted P. aeruginosa is further modulated during interaction with S. aureus and facilitate a proto-cooperative P. aeruginosa–S. aureus relationship

Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner

MexEF-OprN Efflux Pump Exports the Pseudomonas Quinolone Signal (PQS) Precursor HHQ (4-hydroxy-2-heptylquinoline)

Bacterial cells have evolved the capacity to communicate between each other via small diffusible chemical signals termed autoinducers. Pseudomonas aeruginosa is an opportunistic pathogen involved, among others, in cystic fibrosis complications. Virulence of P. aeruginosa relies on its ability to produce a number of autoinducers, including 4-hydroxy-2-alkylquinolines (HAQ). In a cell density-dependent manner, accumulated signals induce the expression of multiple targets, especially virulence factors. This phenomenon, called quorum sensing, promotes bacterial capacity to cause disease. Furthermore, P. aeruginosa possesses many multidrug efflux pumps conferring adaptive resistance to antibiotics. Activity of some of these efflux pumps also influences quorum sensing. The present study demonstrates that the MexEF-OprN efflux pump modulates quorum sensing through secretion of a signalling molecule belonging to the HAQ family. Moreover, activation of MexEF-OprN reduces virulence factor expression and swarming motility. Since MexEF-OprN can be activated in infected hosts even in the absence of antibiotic selective pressure, it could promote establishment of chronic infections in the lungs of people suffering from cystic fibrosis, thus diminishing the immune response to virulence factors. Therapeutic drugs that affect multidrug efflux pumps and HAQ-mediated quorum sensing would be valuable tools to shut down bacterial virulence