Oligomerization of Bacterially Expressed H1N1 Recombinant Hemagglutinin Contributes to Protection Against Viral Challenge

Vaccination is the most effective intervention to prevent influenza and control the spread of the virus. Alternatives are needed to the traditional egg-based vaccine strategy for a more rapid response to new outbreaks. Two different hemagglutinin (HA) fragments (rHA11-326 and rHA153-269) derived from influenza A virus subtype H1N1 were expressed in Escherichia coli and characterized by immunoblot, gel filtration, hemagglutination, and competitive binding assays. rHA11-326 included neutralizing epitopes and the trimerization domain, whereas rHA153-269 included only the head of HA with the neutralizing epitopes. Mice were immunized with rHA11-326 or rHA153-269, and sera were tested for the presence of neutralizing antibodies. Mice were then challenged with H1N1 and infection severity was monitored. rHA11-326 trimerized, whereas rHA153-269 was unable to form oligomers. Both rHA11-326 and rHA153-269 elicited the production of neutralizing antibodies, but only oligomerized rHA11-326 protected against live virus challenges in mice. This study demonstrated that bacterially expressed HA was capable of folding properly and eliciting the production of neutralizing antibodies, and that HA oligomerization contributed to protection against viral challenge. Therefore, prokaryotic-derived vaccine platforms can provide antigenic and structural requirements for viral protection, as well as allow for the rapid and cost-effective incorporation of multiple antigens for broader protection

Preliminary X-ray diffraction studies of the transcriptional inhibitory antibody Fab41.4

The binding of transcription factor ATF-1 to DNA contributes to gene expression and regulation of cell growth. Antibody Mab41.4, raised against ATF-1, and its derivatives Fab41.4 and scFv41.4 inhibit specific DNA binding in vitro and induce apoptotic death of tumor cells in vivo. Structural studies of Fab41.4 were performed to gain insight into the mechanism of action of this potentially therapeutic antibody. The optimal conditions for crystallization of Fab41.4 were determined. Crystals were needle-like in appearance, displayed C2 space-group symmetry and diffracted to a resolution of 1.6 Å. The unit-cell parameters were determined to be a = 186.64, b = 40.22, c = 55.58 Å, α = γ = 90, β = 96.93°. The data set was 97.7% complete. Molecular replacement was performed, resulting in an R value of 44.6%

Preliminary X-ray diffraction studies of the transcriptional inhibitory antibody Fab41.4

The binding of transcription factor ATF-1 to DNA contributes to gene expression and regulation of cell growth. Antibody Mab41.4, raised against ATF-1, and its derivatives Fab41.4 and scFv41.4 inhibit specific DNA binding in vitro and induce apoptotic death of tumor cells in vivo. Structural studies of Fab41.4 were performed to gain insight into the mechanism of action of this potentially therapeutic antibody. The optimal conditions for crystallization of Fab41.4 were determined. Crystals were needle-like in appearance, displayed C2 space-group symmetry and diffracted to a resolution of 1.6 Å. The unit-cell parameters were determined to be a = 186.64, b = 40.22, c = 55.58 Å, α = γ = 90, β = 96.93°. The data set was 97.7% complete. Molecular replacement was performed, resulting in an R value of 44.6%

Arginine Catabolism and Polyamine Biosynthesis Pathway Disparities Within \u3ci\u3eFrancisella tularensis\u3c/i\u3e Subpopulations

Francisella tularensis is a highly infectious zoonotic pathogen with as few as 10 organisms causing tularemia, a disease that is fatal if untreated. Although F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B) share over 99.5% average nucleotide identity, notable differences exist in genomic organization and pathogenicity. The type A clade has been further divided into subtypes A.I and A.II, with A.I strains being recognized as some of the most virulent bacterial pathogens known. In this study, we report on major disparities that exist between the F. tularensis subpopulations in arginine catabolism and subsequent polyamine biosynthesis. The genes involved in these pathways include the speHEA and aguAB operons, along with metK. In the hypervirulent F. tularensis A.I clade, such as the A.I prototype strain SCHU S4, these genes were found to be intact and highly transcribed. In contrast, both subtype A.II and type B strains have a truncated speA gene, while the type B clade also has a disrupted aguA and truncated aguB. Ablation of the chromosomal speE gene that encodes a spermidine synthase reduced subtype A.I SCHU S4 growth rate, whereas the growth rate of type B LVS was enhanced. These results demonstrate that spermine synthase SpeE promotes faster replication in the F. tularensis A.I clade, whereas type B strains do not rely on this enzyme for in vitro fitness. Our ongoing studies on amino acid and polyamine flux within hypervirulent A.I strains should provide a better understanding of the factors that contribute to F. tularensis pathogenicity

Effectiveness of Three Decontamination Treatments against Influenza Virus Applied to Filtering Facepiece Respirators

Filtering facepiece respirators (FFRs) are recommended for use as precautions against airborne pathogenic microorganisms; however, during pandemics demand for FFRs may far exceed availability. Reuse of FFRs following decontamination has been proposed but few reported studies have addressed the feasibility. Concerns regarding biocidal efficacy, respirator performance post decontamination, decontamination cost, and user safety have impeded adoption of reuse measures. This study examined the effectiveness of three energetic decontamination methods [ultraviolet germicidal irradiation (UVGI), microwave-generated steam, and moist heat] on two National Institute for Occupational Safety and Health-certified N95 FFRs (3M models 1860s and 1870) contaminated with H5N1. An aerosol settling chamber was used to apply virus-laden droplets to FFRs in a method designed to simulate respiratory deposition of droplets onto surfaces. When FFRs were examined post decontamination by viral culture, all three decontamination methods were effective, reducing virus load by \u3e4 log median tissue culture infective dose. Analysis of treated FFRs using a quantitative molecular amplification assay (quantitative real-time polymerase chain reaction) indicated that UVGI decontamination resulted in lower levels of detectable viral RNA than the other two methods. Filter performance was evaluated before and after decontamination using a 1% NaCl aerosol. As all FFRs displayed \u3c5%penetration by 300-nm particles, no profound reduction in filtration performance was caused in the FFRs tested by exposure to virus and subsequent decontamination by the methods used. These findings indicate that, when properly implemented, these methods effectively decontaminate H5N1 on the two FFR models tested and do not drastically affect their filtering function; however, other considerations may influence decisions to reuse FFRs

Inhibition of phosphorylated c-Met in rhabdomyosarcoma cell lines by a small molecule inhibitor SU11274

<p>Abstract</p> <p>Background</p> <p>c-Met is a receptor tyrosine kinase (RTK) that is over-expressed in a variety of cancers and involved in cell growth, invasion, metastasis and angiogenesis. In this study, we investigated the role of c-Met in rhabdomyosarcoma (RMS) using its small molecule inhibitor SU11274, which has been hypothesized to be a potential therapeutic target for RMS.</p> <p>Methods</p> <p>The expression level of phosphorylated c-Met in RMS cell lines (RD, CW9019 and RH30) and tumor tissues was assessed by phospho-RTK array and immunohistochemistry, respectively. The inhibition effects of SU11274 on RMS cells were studied with regard to intracellular signaling, cell proliferation, cell cycle and cell migration.</p> <p>Results</p> <p>A high level of phosphorylated c-Met was detected in 2 alveolar RMS cell lines (CW9019 and RH30) and 14 out of 24 RMS tissue samples, whereas relatively low levels of phospho-c-Met were observed in the embryonic RMS cell line (RD). The small molecule SU11274 could significantly reduce the phosphorylation of c-Met, resulting in inhibition of cell proliferation, G1 phase arrest of cell cycle and blocking of cell migration in CW9019 and RH30 cell lines.</p> <p>Conclusion</p> <p>These results might support the role of c-Met in the development and progression of RMS. Furthermore, the inhibitor of c-Met, SU11274, could be an effective targeting therapy reagent for RMS, especially alveolar RMS.</p

Ceftriaxone-Resistant Salmonella Infection Acquired by a Child from Cattle

Background The emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. Resistance to ceftriaxone and the fluoroquinolones, which are used to treat invasive salmonella infections, is rare in the United States. We analyzed the molecular characteristics of a ceftriaxone-resistant strain of Salmonella enterica serotype typhimurium isolated from a 12-year-old boy with fever, abdominal pain, and diarrhea. Methods We used pulsed-field gel electrophoresis and analysis of plasmids and β-lactamases to compare the ceftriaxone-resistant S. enterica serotype typhimurium from the child with four isolates of this strain obtained from cattle during a local outbreak of salmonellosis. Results The ceftriaxone-resistant isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftriaxone. Both ceftriaxone-resistant isolates were resistant to 13 antimicrobial agents; all but one of the resistance determinants were on a conjugative plasmid of 160 kb that encoded the functional group 1 β-lactamase CMY-2. Both ceftriaxone-resistant isolates were closely related to the three other salmonella isolates obtained from cattle, all of which were susceptible to ceftriaxone. Conclusions This study provides additional evidence that antibiotic-resistant strains of salmonella in the United States evolve primarily in livestock. Resistance to ceftriaxone, the drug of choice for invasive salmonella disease, is a public health concern, especially with respect to children, since fluoroquinolones, which can also be used to treat this disease, are not approved for use in children

Large Direct Repeats Flank Genomic Rearrangements between a New Clinical Isolate of Francisella tularensis subsp. tularensis A1 and Schu S4

Francisella tularensis subspecies tularensis consists of two separate populations A1 and A2. This report describes the complete genome sequence of NE061598, an F. tularensis subspecies tularensis A1 isolated in 1998 from a human with clinical disease in Nebraska, United States of America. The genome sequence was compared to Schu S4, an F. tularensis subspecies tularensis A1a strain originally isolated in Ohio in 1941. It was determined that there were 25 nucleotide polymorphisms (22 SNPs and 3 indels) between Schu S4 and NE061598; two of these polymorphisms were in potential virulence loci. Pulsed-field gel electrophoresis analysis demonstrated that NE061598 was an A1a genotype. Other differences included repeat sequences (n = 11 separate loci), four of which were contained in coding sequences, and an inversion and rearrangement probably mediated by insertion sequences and the previously identified direct repeats I, II, and III. Five new variable-number tandem repeats were identified; three of these five were unique in NE061598 compared to Schu S4. Importantly, there was no gene loss or gain identified between NE061598 and Schu S4. Interpretation of these data suggests there is significant sequence conservation and chromosomal synteny within the A1 population. Further studies are needed to determine the biological properties driving the selective pressure that maintains the chromosomal structure of this monomorphic pathogen