Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination-reinitiation mechanism

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level

Longitudinal microarray analysis of cell surface antigens on peripheral blood mononuclear cells from HIV+ individuals on highly active antiretroviral therapy

<p>Abstract</p> <p>Background</p> <p>The efficacy of highly active antiretroviral therapy (HAART) determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6) achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6) responded intermittently. Blood samples were collected over an average of 3 years and 5–8 time points were selected for microarray assay and statistical analysis.</p> <p>Results</p> <p>Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28) for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33) were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively.</p> <p>Conclusion</p> <p>Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.</p

Characterization of an Axially Sampling Time-of-flight Mass Spectrometer for Upper Atmospheric Measurements

The mesosphere/lower thermosphere (MLT) lies between the turbulent mixing and diffusive layers of the earth’s upper atmosphere. Temperatures in this region are varied and include the coldest region of the earth’s atmosphere, the mesopause. Too high for aircraft and too low for satellites, the only method of direct access to the MLT is by sounding rocket for periods of at most a few minutes. Because of this, the MLT is the most difficult region of the earth’s atmosphere to access and is therefore the least understood region of the earth’s atmosphere. Accurate in-situ measurements of MLT species are important for the following reasons: •CO2 concentration profiles collected in-situ will be useful in validating and improving atmospheric temperature measurements made by satellite based instruments, for example, the Sounding of the Atmosphere using Broadband Emission Radiometry on the TIMED spacecraft. • Study the transport of atmospheric species near the turbopause region. For example, NO, which is thought to be linked to ozone depletion in the stratosphere • In-situ concentration data will help to improve atmospheric models, such as the MSIS model We present an axially-sampling time-of-flight mass spectrometer (TOF-MS) suitable for deployment aboard sounding rockets to make measurements in the MLT. Use of a Bradbury- Nielsen gate to modulate ions makes on-axis sampling possible. The TOF-MS also employs a pressure tolerant microchannel plate (MCP) detector capable of operating at pressures into the 10-4 torr range. We have built and are currently testing a prototype instrument in our ion optics facility. Experiments to date demonstrate the potential of the TOF-MS to successfully make measurements in the MLT and thereby improve our knowledge of this important region of the earth’s atmosphere

A New Mass Spectrometer for Upper Atmospheric Measurements in the Auroral Region

We have previously presented a new rocket-borne time-of-flight mass spectrometer (TOF-MS) for measurements in the mesosphere / lower thermosphere (MLT). Traditionally, mass spectrometry in the MLT has been difficult, mainly due to the elevated ambient pressures of the MLT and high speeds of a sounding rocket flight, which affect the direct sampling of the ambient atmosphere and spatial resolution. The TOF-MS is a versatile, inherently adaptable, axial-sampling instrument, capable of operating in a traditional TOF mode or in a multiplexing Hadamard-transform mode where high spatial resolution is desired. To minimize bow shock effects at low altitudes (~70-110km), the ram surface of the TOF-MS can be cryogenically cooled using liquid He to adsorb impinging gas particles. The vacuum pumping system for the TOF-MS is tailored to the specific mission and instrument configuration. Depending on the instrument gas load and operating altitude, cryo, miniature turbo pump or getter-based pumping systems may be employed. Terrestrial TOF-MS instruments often employ a reflectron, essentially an ion-mirror, to improve mass resolving power and compensate for the thermal velocity distribution of particles being measured. The TOF-MS can be arranged in either a simple linear or reflectron configuration. Simulations and modeling are used to compare instrument mass resolution for linear and reflectron configurations for several variable conditions including vehicle velocity and ambient temperature, ultimately demonstrating the potential to make rocket-borne mass spectrometry measurements with unit-mass resolution up to at least 48 amu. Preliminary analyses suggest that many species of interest (including He, CO2, O2, O2 , N2, N2 , and NO ) can be measured with an uncertainty below 10% relative standard deviation on a sounding rocket flight. We also present experimental data for a laboratory prototype linear TOF-MS. Experimental data is compared to simulation and modeling efforts to validate and confirm instrument performance and capability. Two proposed rocket campaigns for investigations of the auroral region include the TOF-MS. By making accurate composition measurements of the neutral atmosphere from 70 to 120km, Mass Spectrometry of the Turbopause Region (MSTR) aims to improve the accuracy of temperature measurements in the turbopause region, improve the MSIS model atmosphere and examine the transition from the turbulently mixed lower atmosphere to the diffusive equilibrium of the upper atmosphere. The ROCKet-borne STorm Energetics of Auroral Dosing in the E-region (ROCK-STEADE) mission will study energy transfer in the E-region during an aurora by examining auroral emissions and measuring concentrations of neutrals and ions. The instrument suite for ROCK-STEADE includes two mass spectrometers, one each to measure neutrals and ions in the altitude range of 70 - 170km. The ability of the TOF-MS instrument to make accurate measurements will greatly aid in better understanding the MLT

Mechanisms of HIV non-progression; robust and sustained CD4+ T-cell proliferative responses to p24 antigen correlate with control of viraemia and lack of disease progression after long-term transfusion-acquired HIV-1 infection

<p>Abstract</p> <p>Background</p> <p>Elite non-progressors (plasma viral load <50 copies/ml while antiretroviral naive) constitute a tiny fraction of HIV-infected individuals. After 12 years follow-up of a cohort of 13 long-term non-progressors (LTNP) identified from 135 individuals with transfusion-acquired HIV infection, 5 remained LTNP after 23 to 26 years infection, but only 3 retained elite LTNP status. We examined the mechanisms that differentiated delayed progressors from LTNP in this cohort.</p> <p>Results</p> <p>A survival advantage was conferred on 12 of 13 subjects, who had at least one host genetic factor (HLA, chemokine receptor or TLR polymorphisms) or viral attenuating factor (defective <it>nef</it>) associated with slow progression. However, antiviral immune responses differentiated the course of disease into and beyond the second decade of infection. A stable p24-specific proliferative response was associated with control of viraemia and retention of non-progressor status, but this p24 response was absent or declined in viraemic subjects. Strong Gag-dominant cytotoxic T lymphocyte (CTL) responses were identified in most LTNP, or Pol dominant-CTL in those with <it>nef</it>-defective HIV infection. CTL were associated with control of viraemia when combined with p24 proliferative responses. However, CTL did not prevent late disease progression. Individuals with sustained viral suppression had CTL recognising numerous Gag epitopes, while strong but restricted responses to one or two immunodominant epitopes was effective for some time, but failed to contain viraemia over the course of this study. Viral escape mutants at a HLA B27-restricted Gag-p24 epitope were detected in only 1 of 3 individuals, whereas declining or negative p24 proliferative responses occurred in all 3 concurrent with an increase in viraemia.</p> <p>Conclusion</p> <p>Detectable viraemia at study entry was predictive of loss of LTNP status and/or disease progression in 6 of 8, and differentiated slow progressors from elite LTNP who retained potent virological control. Sustained immunological suppression of viraemia was independently associated with preserved p24 proliferative responses, regardless of the strength and breadth of the CTL response. A decline in this protective p24 response preceded or correlated with loss of non-progressor status and/or signs of disease progression.</p

Fibrinogen in traumatic haemorrhage: A narrative review

Haemorrhage in the setting of severe trauma is associated with significant morbidity and mortality. There is increasing awareness of the important role fibrinogen plays in traumatic haemorrhage. Fibrinogen levels fall precipitously in severe trauma and the resultant hypofibrinogenaemia is associated with poor outcomes. Hence, it has been postulated that early fibrinogen replacement in severe traumatic haemorrhage may improve outcomes, although, to date there is a paucity of high quality evidence to support this hypothesis. In addition there is controversy regarding the optimal method for fibrinogen supplementation. We review the current evidence regarding the role of fibrinogen in trauma, the rationale behind fibrinogen supplementation and discuss current research.Griffith Health, School of Medical ScienceNo Full Tex

Integrated supercritical fluid extraction and bioprocessing

ABSTRACT Supercritical fluids are increasingly being used and promoted at a laboratory and pilot scale to produce high value, natural bioactives from biologically based raw materials. Supercritical CO 2 is overwhelmingly the solvent of choice for these operations, but is largely limited to the processing of dry raw materials and the extraction of low polarity, low molecular weight compounds. The use of co-solvents and the use of alternative &apos;near-critical&apos; extraction fluids such as dimethyl ether show potential to mitigate these limitations. Commercialisation of new supercritical extraction processes has arguably been limited because the supercritical extraction process has been developed in isolation of other processing steps necessary to achieve a successful product. This study reviews recent developments in integrated processing that incorporate the use of supercritical fluids for bioseparations and in particular process schemes that produce high value natural bioactives. Integrated processes include prior operation (fermentation, extraction, enzyme pre-treatment, physical fractionation or size reduction) followed by supercritical extraction or fractionation and processes in which operations are carried out in situ in supercritical fluids (supercritical chromatography, enzymatic conversion, precipitation and coating of solutes). The use of co-solvents and alternative extraction solvents in these processes is discussed. Prospects for future developments are also discussed