A Strain Gauge Monitor (SGM) for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH.

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response to diel-cycling hypoxia. A Wheatstone bridge was connected to strain gauges that were attached to the shells of oysters (Crassostrea virginica). The recorded signals allowed for the opening and closing of the bivalves to be recorded continuously over two-day periods of experimentally-induced diel-cycling hypoxia and diel-cycling changes in pH. Here, we describe a protocol for developing an inexpensive strain gauge monitor and describe, in an example laboratory experiment, how we used it to measure the valve gape behavior of Eastern oysters (C. virginica), in response to diel-cycling hypoxia and cyclical changes in pH. Valve gape was measured on oysters subjected to cyclical severe hypoxic (0.6 mg/L) dissolved oxygen conditions with and without cyclical changes in pH, cyclical mild hypoxic (1.7 mg/L) conditions and normoxic (7.3 mg/L) conditions. We demonstrate that when oysters encounter repeated diel cycles, they rapidly close their shells in response to severe hypoxia and close with a time lag to mild hypoxia. When normoxia is restored, they rapidly open again. Oysters did not respond to cyclical pH conditions superimposed on diel cycling severe hypoxia. At reduced oxygen conditions, more than one third of the oysters closed simultaneously. We demonstrate that oysters respond to diel-cycling hypoxia, which must be considered when assessing the behavior of bivalves to dissolved oxygen. The valve SGM can be used to assess responses of bivalve molluscs to changes in dissolved oxygen or contaminants. Sealing techniques to better seal the valve gape strain gauges from sea water need further improvement to increase the longevity of the sensors.

mRNA vaccines - a new era in vaccinology.

mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.

Design and on-orbit operation of the Soft X-ray Spectrometer ADR on the Hitomi Observatory.

The Soft X-ray Spectrometer (SXS) instrument that flew on the Astro-H observatory was designed to perform imaging and spectroscopy of x-rays in the energy range of 0.2 to 13 keV with a resolution requirement of 7 eV or better. This was accomplished using a 6x6 array of x-ray microcalorimeters cooled to an operating temperature of 50 mK by an adiabatic demagnetization refrigerator (ADR). The ADR consisted of three stages in order to operate using either a 1.2 K superfluid helium bath or a 4.5 K Joule-Thomson (JT) cryocooler as its heat sink. The design was based on the following operating strategy. After launch, while liquid helium was present (cryogen mode), two of the ADR's stages would be used to single-shot cool the detectors, using the helium as a heat sink. When the helium was eventually depleted (cryogen-free mode), all three ADR stages would be used to continuously cool the helium tank to about 1.5 K, and to single-shot cool the detectors (to 50 mK), using the JT cryocooler as a heat sink. The Astro-H observatory, renamed Hitomi after its successful launch in February 2016, carried approximately 36 liters of helium into orbit. Based on measurements during ground testing, the average heat load on the helium was projected to be 0.66 mW, giving a lifetime of more than 4 years. On day 5, the helium had cooled to <1.4 K and ADR operation began, successfully cooling the detector array to 50 mK. The ADR's hold time steadily increased to 48 hours as the helium cooled to a temperature of 1.12 K. As the commissioning phase progressed, the ADR was recycled (requiring approximately 45 minutes) periodically, either in preparation for science observations or whenever the 50 mK stage approached the end of its hold time. In total, 18 cycles were completed by the time an attitude control anomaly led to an unrecoverable failure of the satellite on day 38. This paper presents the design, operation and on-orbit performance of the ADR in cryogen mode as the foreshortened mission did not provide an opportunity to test cryogen-free mode.

Comprehensive Characterization of Reference Standard Lots of HIV-1 Subtype C Gp120 Proteins for Clinical Trials in Southern African Regions.

Two HIV-1 subtype C gp120 protein candidates were the selected antigens for several experimental vaccine regimens now under evaluation in HVTN 100 Phase I/II clinical trial aiming to support the start of the HVTN 702 Phase IIb/III trial in southern Africa, which is designed to confirm and extend the partial protection seen against HIV-1 infection in the RV144 Thai trial. Here, we report the comprehensive physicochemical characterization of the gp120 reference materials that are representative of the clinical trial materials. Gp120 proteins were stably expressed in Chinese Hamster Ovary (CHO) cells and subsequently purified and formulated. A panel of analytical techniques was used to characterize the physicochemical properties of the two protein molecules. When formulated in the AS01 Adjuvant System, the bivalent subtype C gp120 antigens elicited 1086.C- and TV1.C-specific binding antibody and CD4+ T cell responses in mice. All the characteristics were highly representative of the Clinical Trial Materials (CTM). Data from this report demonstrate the immunogenicity of the gp120 antigens, provide comprehensive characterization of the molecules, set the benchmark for assessment of current and future CTM lots, and lay the physicochemical groundwork for interpretation of future clinical trial data.

Operating Modes and Cooling Capabilities of the 3-Stage ADR Developed for the Soft-X-ray Spectrometer Instrument on Astro-H.

A 3-stage adiabatic demagnetization refrigerator (ADR)[1] is used on the Soft X-ray Spectrometer instrument[2] on Astro-H[3] to cool a 6×6 array of x-ray microcalorimeters to 50 mK. The ADR is supported by a cryogenic system[4] consisting of a superfluid helium tank, a 4.5 K Joule-Thomson (JT) cryocooler, and additional 2-stage Stirling cryocoolers that pre-cool the JT cooler and cool radiation shields within the cryostat. The ADR is configured so that it can use either the liquid helium or the JT cryocooler as its heat sink, giving the instrument an unusual degree of tolerance for component failures or degradation in the cryogenic system. The flight detector assembly, ADR and dewar were integrated into the flight dewar in early 2014, and have since been extensively characterized and calibrated. This paper summarizes the operation and performance of the ADR in all of its operating modes.

Nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases.

Cardioviruses disrupt nucleocytoplasmic transport through the activity of their leader (L) protein. We have shown that hyperphosphorylation of nuclear pore proteins (nucleoporins or Nups), including Nup62, Nup153, and Nup214, is central to this L protein function and requires one or more cytosolic kinases. In this study, potential cellular enzymes involved in encephalomyocarditis virus (EMCV) L-directed Nup phosphorylation were screened with a panel of specific, cell-permeating kinase inhibitors. Extracellular signal-regulated receptor kinase (ERK) and p38 mitogen-activated protein kinase inhibitors (U0126 and SB203580) were sufficient to block Nup hyperphosphorylation in EMCV-infected or L-expressing cells. Recombinant L alone, in the absence of infection, triggered activation of ERK and p38, independent of their upstream signaling cascades. Conserved residues within the L zinc finger (Cys(19)) and acidic domain (Asp(48),(51),(52),(55)) were essential for this activation and for the phosphorylation of Nups, suggesting that the phenomena are linked. Analysis of the hyperphosphorylated Nup species revealed only phosphoserine and phosphothreonine residues. The sizes of the tryptic phosphopeptides derived from Nup62 were compatible with sites in the Phe/Gly repeat domain which display common consensus sequences for ERK and p38 substrates. The results provide strong evidence that ERK and p38 are the probable effector kinases required for L-dependent inhibition of nuclear trafficking.

Leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses.

Picornaviruses disrupt nucleocytoplasmic trafficking pathways during infection. Poliovirus and rhinovirus inhibit nuclear protein import/export through a series of 2A protease-dependent cleavages within nuclear pore proteins (nucleoporins [Nups]), including Nup62, Nup98, and Nup153. Cardioviruses lack the same protease and instead affect trafficking inhibition through an activity mapped to their leader (L) protein, a 67- to 76-amino acid (aa) polypeptide with no known enzymatic activity. We have shown that L from encephalomyocarditis virus (EMCV) binds and inhibits the activity of Ran-GTPase, a key regulator of nucleocytoplasmic transport. We now report that recombinant EMCV L triggers the unregulated efflux of protein cargo from preloaded HeLa cell nuclei in cell-free reactions dependent upon Xenopus egg cytosol or HeLa cell-derived cytosol. Recombinant L was the only viral protein necessary for this activity or for nuclear protein import inhibition. Mutational disruption of the L protein zinc finger domain (C(19)A) abrogated the inhibitory activity for both import and efflux in cell extracts, but mutations in the C-terminal acidic domain of L (aa 37 to 61) did not. Notably, HeLa cell nuclei treated with L, or those from EMCV-infected cells, showed reproducibly altered patterns of nucleoporin phosphorylation. Nup62, Nup153, and Nup214 each became hyperphosphorylated in an L-dependent manner. Staurosporine, a broad-spectrum kinase inhibitor, blocked this phosphorylation and rescued nuclear import/export activity from L-dependent inhibition. Therefore, cardioviruses target the same group of nucleoporins as enteroviruses, but the effector mechanism triggered by L (or L-Ran complexes) involves a unique cytosol-dependent phosphorylation cascade rather than proteolysis.

Performance of the EBIT calorimeter spectrometer.

The EBIT calorimeter spectrometer (ECS) is a new high-resolution, broadband x-ray spectrometer that has recently been installed at the Electron Beam Ion Trap Facility (EBIT) at the Lawrence Livermore National Laboratory. The ECS is an entirely new production class spectrometer that replaces the XRS/EBIT spectrometer that has been operating at EBIT since 2000. The ECS utilizes a 32-pixel x-ray calorimeter array from the XRS instrument on the Suzaku x-ray observatory. Eighteen of the pixels are optimized for the 0.1-10 keV band and yield 4.5 eV full width at half maximum energy resolution and 95% quantum efficiency at 6 keV. In addition, the ECS includes 14 detector pixels that are optimized for the high-energy band with a bandpass from 0.5 to over 100 keV with 34 eV resolution and 32% quantum efficiency at 60 keV. The ECS detector array is operated at 50 mK using a five stage cryogenic system that is entirely automated. The instrument takes data continuously for over 65 h with a 2.5 h recycle time. The ECS is a nondispersive, broadband, highly efficient spectrometer that is one of the prime instruments at the EBIT facility. The instrument is used for studies of absolute cross sections, charge exchange recombination, and x-ray emission from nonequilibrium plasmas, among other measurements in our laboratory astrophysics program.

NMR structure of the mengovirus Leader protein zinc-finger domain.

The Leader protein is a defining feature of picornaviruses from the Cardiovirus genus. This protein was recently shown to inhibit cellular nucleocytoplasmic transport through an activity mapped to its zinc-binding region. Here we report the three-dimensional solution structure determined by nuclear magnetic resonance (NMR) spectroscopy of this domain (residues 5-28) from mengovirus. The domain forms a CHCC zinc-finger with a fold comprising a beta-hairpin followed by a short alpha-helix that can adopt two different conformations. This structure is divergent from those of other eukaryotic zinc-fingers and instead resembles motifs found in a group of DNA-binding proteins from Archaea.

A picornavirus protein interacts with Ran-GTPase and disrupts nucleocytoplasmic transport.

Active nucleocytoplasmic transport of protein and RNA in eukaryotes depends on the Ran-GTPase system to regulate cargo-receptor interactions. Several viruses, including the RNA picornaviruses, encode factors that alter nuclear transport with the aim of suppressing synthesis of antiviral factors and promoting viral replication. Picornaviruses in the cardiovirus genus express a unique 67-aa Leader protein (L), known to alter the subcellular distribution of IFN regulatory proteins targeted to the nucleus. We report here that L binds directly to Ran and blocks nuclear export of new mRNAs. In Xenopus egg extracts, recombinant L also inhibits mitotic spindle assembly, a RanGTP function crucial to cell-cycle progression. We propose that L inhibits nucleocytoplasmic transport during infection by disrupting the RanGDP/GTP gradient. This inhibition triggers an efflux of nuclear proteins necessary for viral replication and causes IFN suppression. To our knowledge, L is the first viral picornaviral protein to interact directly with Ran and modulate the Ran-dependent nucleocytoplasmic pathway.