Development and pre-clinical evaluation of two LAIV strains against potentially pandemic H2N2 influenza virus.

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.

A view on phosphate ester photochemistry by time-resolved solid state NMR. Intramolecular redox reaction of caged ATP.

The light-driven intramolecular redox reaction of adenosine-5'-triphosphate-[P3-(1-(2-nitrophenyl)-ethyl)]ester (caged ATP) has been studied in frozen aqueous solution using time-resolved solid state NMR spectroscopy under continuous illumination conditions. Cleavage of the phosphate ester bond leads to 0.3, 1.36, and 6.06 ppm downfield shifts of the alpha-, beta-, and gamma-phosphorus resonances of caged ATP, respectively. The observed rate of ATP formation is 2.4 +/- 0.2 h(-1) at 245 K. The proton released in the reaction binds to the triphosphate moiety of the nascent ATP, causing the upfield shifts of the 31P resonances. Analyses of the reaction kinetics indicate that bond cleavage and proton release are two sequential processes in the solid state, suggesting that the 1-hydroxy,1-(2-nitrosophenyl)-ethyl carbocation intermediate is involved in the reaction. The beta-phosphate oxygen atom of ATP is protonated first, indicating its proximity to the reaction center, possibly within hydrogen bonding distance. The residual linewidth kinetics are interpreted in terms of chemical exchange processes, hydrogen bonding of the beta-phosphate oxygen atom and evolution of the hydrolytic equilibrium at the triphosphate moiety of the nascent ATP. Photoreaction of caged ATP in situ gives an opportunity to study structural kinetics and catalysis of ATP-dependent enzymes by NMR spectroscopy in rotating solids.

Haloalkaliphilic diazotrophs in soda solonchak soils.

Nitrogen fixation (NF) potential was measured in more than 40 samples of soda solonchak soils with the pH of water extract between 9.5 and 11.0 collected in several locations of Central Asia and in Egypt, using the acetylene reduction method. NF was detected in most of the samples. Maximal rates were observed under microaerophilic-anaerobic conditions with glucose as a substrate. In most cases, the NF negatively correlated with salt content and alkalinity. Five enrichments at pH 10 under micro-oxic conditions with glucose resulted in stable haloalkaliphilic mixed cultures, with diazotrophic component(s) active up to 2.0-3.0 M total Na(+). The cultures were dominated by Gram-positive spore-forming bacteria. Molecular cloning of nifH genes demonstrated the presence of two phylogenetic lineages of diazotrophs in the enrichments affiliated with the low-GC Gram-positive bacteria (in rRNA groups 1 and 6 of bacilli and in Clostridiales). Isolation of pure cultures of haloalkaliphilic diazotrophs from micro-oxic enrichments yielded nine strains, comprising two phylogenetic lineages. Most of the isolates (eight) were affiliated with the aerotolerant fermentative haloalkaliphilic bacterium Amphibacillus tropicus and a single strain clustered with the obligately anaerobic haloalkaliphile Bacillus arseniciselenatis. Diazotrophy has never been recognized previously in these groups of Gram-positive bacteria. Overall, the results demonstrated the existence, in soda solonchak soils, of a novel group of free-living fermentative diazotrophic bacteria active at extremely haloalkaline conditions.

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase.

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD(+) and assistance of a divalent metal cofactor Mg(2+). Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover (31)P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg(2+), and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.

Association of CD2AP with dynamic actin on vesicles in podocytes.

The docking protein CD2AP (CD2-associated protein) serves a nonredundant function in podocytes as CD2AP knockout mice die of renal failure at the age of 6-7 wk. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Although CD2AP has been shown to interact with proteins regulating actin polymerization, with proteins of the slit diaphragm, and with the endocytic machinery, its critical function in podocytes remains unclear. In conditionally immortalized mouse podocytes, we demonstrate that CD2AP colocalizes with cortactin and F-actin in spots of < or =0.5-microm diameter. Confocal time-lapse microscopy in living podocytes expressing GFP-CD2AP or GFP-actin revealed that spots are motile, possess a limited lifetime, and are frequently associated with vesicles. A significant portion of spot-associated vesicles belongs to a later endosomal-sorting compartment, characterized by delayed uptake of fluorescent dextran (10 kDa) and by colocalization with Rab4, but not Rab5 and AP-2. Rapid accumulation of microinjected G-actin in spots and abrogation of spot motility by jasplakinolide demonstrate that spot movements depend on actin polymerization. Furthermore, a high turnover (half-time < 10 s) of CD2AP in spots was demonstrated by FRAP (fluorescence recovery after photobleaching). Our results demonstrate that CD2AP is associated with dynamic actin in a specific late endosomal compartment in podocytes, suggesting that CD2AP might be crucially involved in endosomal sorting and/or trafficking via regulation of actin assembly on vesicles.