Automatic classification of spectra from the Infrared Astronomical Satellite (IRAS)

A new classification of Infrared spectra collected by the Infrared Astronomical Satellite (IRAS) is presented. The spectral classes were discovered automatically by a program called Auto Class 2. This program is a method for discovering (inducing) classes from a data base, utilizing a Bayesian probability approach. These classes can be used to give insight into the patterns that occur in the particular domain, in this case, infrared astronomical spectroscopy. The classified spectra are the entire Low Resolution Spectra (LRS) Atlas of 5,425 sources. There are seventy-seven classes in this classification and these in turn were meta-classified to produce nine meta-classes. The classification is presented as spectral plots, IRAS color-color plots, galactic distribution plots and class commentaries. Cross-reference tables, listing the sources by IRAS name and by Auto Class class, are also given. These classes show some of the well known classes, such as the black-body class, and silicate emission classes, but many other classes were unsuspected, while others show important subtle differences within the well known classes

Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, RT and Gp120 and boosting with recombinant MVA induces a robust T cell response in mice

In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCGΔ panCD ) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCGΔ panCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8 + T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 10 5 cfu to 10 7 cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCGΔ panCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-γ-spot forming cells than mice primed with rBCGΔ panCD containing an empty vector control. Priming with the individual rBCGΔ panCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4 + and CD8 + T cells producing IFN-γ and TNF-α and CD4 + cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix

Priming with a Recombinant Pantothenate Auxotroph of Mycobacterium bovis BCG and Boosting with MVA Elicits HIV-1 Gag Specific CD8+ T Cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8+ T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4+ T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge

The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10 7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10 6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge

Regulation and characterization of the nitrogen assimilatory gene cluster in Clostridium acetobutylicum P262

The solventogenic Clostridium acetobutylicum strain P262 was used for the commercial production of solvents in South Africa from 1945 to 1983 (Jones and Woods, 1986a). Our laboratory has focused on understanding two: fundamental aspects of its physiology, namely nitrogen metabolism and electron transport pathways. The long-term goal is the potential genetic modification of nitrogen utilization and/or electron distribution in the cell, to manipulate fermentation patterns for improved growth rate and solvent yields. The main aims of this research project were to: extend the sequence analysis of the glnA locus encoding the glutamine synthetase (GS) enzyme (Janssen et al., 1990; Fierro-Monti et al., 1992); to identify and characterize the locus encoding the structural genes for the glutamate synthase (GOGAT) enzyme; and to detennine the mechanisms and growth conditions which regulate the activity of these two key enzymes of nitrogen assimilation. In addition, we were interested in characterizing clone pMET13Cl, which was isolated by a selection system developed to clone genes involved in electron transport from C. acetobutylicum (Santangelo et al., 1991). Sequence analyses revealed that the region downstream of glnA and the putative regulatory gene, glnR (Reid and Woods, 1995), encoded the structural genes for the large (a) and small (J) subunits of GOGAT, respectively. This is the first report in which the genes encoding GS and GOGAT are genetically linked. The a subunit was designated gltA, and the downstream f3 subllll.it was designated gltB. All the likely cofactor and substrate binding sites identified in GOGAT enzymes (Vanoni and Curti, 1999) were conserved in the deduced gltA and gltB polypeptides. The identity of the gltA and gltB genes were functionally confirmed by complementation studies involving their co-expression from separate constructs in an E. coli glutamate auxotroph (strain: Mx:3004), which restored the ability of this mutant to grow with ammonia as the sole source of nitrogen. Physiological studies on the germination, growth and differentiation patterns of C. acetobutylicum P262 were assessed in relation to different nitrogen conditions. Significantly, organic nitrogen (casamino acids) was the preferred source of nitrogen, and not ammonia as ll previously asswned. These studies led to the development of nitrogen limiting conditions (0.025% casamino acids + 0.15% glutamine) and nitrogen rich conditions (0.2% casamino acids) used for regulatory studies. GOG AT activity was optimised. It appeared to be sensitive to oxygen and specific for the co-enzyme NADH. Both GS and GOGAT activities were regulated by the nitrogen source in a similar way: induced in the nitrogen limiting conditions and repressed in the nitrogen rich conditions. Northern blot analyses, in conjunction with the enzyme activity profiles and feedback inhibition studies, indicated that GS and GOGAT activities were regulated primarily at the level of transcription. Furthermore, glnA. andglnR, and gltA and gltB are each transcribed as an operon under nitrogen limiting conditions. No assimilatory GDH activity could be detected. The implications of these results, as well as sequence features identified, are discussed in context with a proposed model for the regulation of GS and GOGAT activity in C. acetobutylicum P262. Analysis of plasmid pMET13Cl identified a gene whose predicted -46 kDa product was associated with an electron transport function. The deduced amino acid sequence was not typical of electron transport proteins, but rather shared striking homology to bacterial GOGAT p subunit polypeptides. This p subunit-like gene was thus designated gltX. We were, however, unable to relate it to GOGAT activity or nitrogen metabolism. Rather, it appears to belong to a novel family of FAD-dependent NAD(P)H oxidoreductases suggested by Vanoni and Curti (1999) and supported by an analysis of the evolutionary relationships of the GOGAT subunits/domains from various sources

The Role of Complement System in Septic Shock

Septic shock is a critical clinical condition with a high mortality rate. A better understanding of the underlying mechanisms is important to develop effective therapies. Basic and clinical studies suggest that activation of complements in the common cascade, for example, complement component 3 (C3) and C5, is involved in the development of septic shock. The involvement of three upstream complement pathways in septic shock is more complicated. Both the classical and alternative pathways appear to be activated in septic shock, but the alternative pathway may be activated earlier than the classical pathway. Activation of these two pathways is essential to clear endotoxin. Recent investigations have shed light on the role of lectin complement pathway in septic shock. Published reports suggest a protective role of mannose-binding lectin (MBL) against sepsis. Our preliminary study of MBL-associated serine protease-2 (MASP-2) in septic shock patients indicated that acute decrease of MASP-2 in the early phase of septic shock might correlate with in-hospital mortality. It is unknown whether excessive activation of these three upstream complement pathways may contribute to the detrimental effects in septic shock. This paper also discusses additional complement-related pathogenic mechanisms and intervention strategies for septic shock

Schematic map and restriction enzyme digests of pHS501, pRC501 and pHS121 plasmid DNA isolated from rBCG.

<p>(<b>A</b>) Schematic map of <i>E. coli</i>/mycobacterial shuttle vector. (<b>B</b>) Lanes 1 & 8 contain the molecular weight marker GeneRuler^TM 1 kb Ladder (Fermentas, S.A.). Lanes 2 & 3 contain pHS501 plasmid DNA. Lanes 4 & 5 contain pRC501 plasmid DNA. Lanes 6 & 7 contain pHS121 plasmid DNA. Lanes 2, 4 & 6 contain plasmid DNA isolated prior to transformation into BCG (positive controls). Lanes 3, 5 & 7 contain plasmid DNA isolated from rBCG. Plasmid DNA in lanes 2 to 7 was digested with restriction enzymes <i>Xba</i>I and <i>Hpa</i>I. Results were the same for rBCG and rBCGΔ<i>panCD</i>. Enzymatic restriction analysis of only 2 representative samples are shown for each rBCG, however plasmid DNA was isolated from a minimum of 20 of each rBCG.</p

Influence of vector on IFN-γ ELISPOT responses to a rBCG-Gag or rBCGpan-Gag prime and rMVA boost.

<p>Groups of mice were primed with the indicated rBCG vaccines (10⁷ cfu or 10⁵ cfu) and then boosted with rMVA (10⁴ pfu) on day 28. One group of mice was left unvaccinated then vaccinated on day 28 with rMVA. On day 40 spleens were harvested and splenocytes pooled from 5 mice per group. (A) IFN-γ ELISPOT assays with Gag CD8⁺ T cells and CD4⁺ T cell peptides or peptide H CD8⁺ T cell peptide. Bars are the average and standard deviation of the average IFN-γ ELISPOT responses for the indicated individual peptides for 3 separate experiments. Asterisks indicate statistical significance of the mean IFN-γ ELISPOT responses for the individual peptides for a rBCG-Gag or rBCGpan-Gag vaccine prime and rMVA boost compared to that for the respective rBCG-Control or rBCGpan-Control vaccine prime and rMVA boost. (B) Splenocytes were stained with H-2K^d and H-2D^d MHC class I pentamers folded with the Gag CD8⁺ T cell peptide or peptide H CD8⁺ T cell peptide and flow cytometry was used to determine the frequency of Gag- and peptide H-specific CD8⁺ T cells in the splenocyte population. Bars are the average of triplicate values for Gag- and peptide H-specific CD8⁺ T cells expressed as a percentage of the total gated CD8⁺ T cell population for a single experiment. The coefficient of variation of all average values (standard deviation of the average expressed as a percentage of the average) was less than 0.01%. Asterisks indicate the statistical significance of the mean values for the percentage of Gag- or peptide H-specific CD8⁺ T cells for a rBCG-Gag or rBCGpan-Gag vaccine prime and rMVA boost compared to that for the respective rBCG-Control or BCGpan-Control vaccine prime and rMVA boost. Respective differences for peptide responses between groups are also indicated. *<0.01; **<0.05; Student's t-test for means of unpaired data.</p

IFN-γ ELISPOT responses induced by a prime with the individual rBCG-HIV vaccines or a mix of the rBCG-HIV vaccines and rMVA boost.

<p>Groups of mice were primed with either the individual rBCGpan-Gag, rBCGpan-RT or rBCGpan-gp120 vaccines (10⁷ cfu) or a rBCG vaccine mix (3×10⁷ cfu) prepared by mixing 10⁷ cfu of the individual rBCGpan-Gag, rBCGpan-RT and rBCGpan-gp120 vaccines. Groups of mice vaccinated with the rBCGpan-Control vaccine (10⁷ cfu to act as control for the individual vaccine vaccinations or 3×10⁷ cfu to act as control for the mix of the individual vaccine vaccinations) served as controls. All groups of mice were then boosted with MVA (10⁴ pfu) on day 28. On day 40 spleens were harvested and splenocytes pooled from 5 mice per group were used in an IFN-γ ELISPOT (<b>A</b>) or IL-2 ELISPOT (<b>B</b>) assay with Gag, RT, Env and peptide H CD8⁺ T cell and CD4⁺ T cell peptides. Bars are the average and standard deviation of the average IFN-γ ELISPOT (A) or IL-2 ELISPOT responses for the indicated individual peptides for 3 separate experiments. Statistical significance (Student's t-test for means of unpaired data) of the mean IFN-γ ELISPOT or IL-2 ELISPOT responses for the individual peptides compared to that for the control is indicated.</p