Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: Biological validation of the proposed HLA A24 supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

Full-scale demonstration of biological nutrient removal in a single tank SBR process

Complete biological nutrient removal (BNR) in a single tank, sequencing batch reactor (SBR) process, is demonstrated here at full-scale on a typical domestic wastewater. The unique feature of the UniFed process is the introduction of the influent into the settled sludge blanket during the settling and decant periods of the SBR operation. This achieves suitable conditions for denitrification and anaerobic phosphate release which is critical to successful biological phosphorus removal, It also achieves a selector effect, which helps in generating a compact, well settling biomass in the reactor. The results of this demonstration show that it is possible to achieve well over 90% removal of GOD, nitrogen and phosphorus in such a process. Effluent quality achieved over a six-month operating period directly after commissioning was: 29 mg/l GOD, 0.5 mg/l NH4-N, 1.5 mg/l NOx-N and 1.5 mg/l PO4-P (50%-iles of daily samples). During an 8-day, intensive sampling period, the effluent BOD5 was

Nitrogen cycling in degraded Leucaena leucocephala-Brachiaria decumbens pastures on an acid infertile soil in south-east Queensland, Australia

A grazing trial was conducted to quantify N cycling in degraded Leucaena leucocephala (leucaena)-Brachiaria decumbens (signal grass) pastures grown on an acid, infertile, podzolic soil in south-east Queensland. Nitrogen accumulation and cycling in leucaena-signal grass pastures were evaluated for 9 weeks until all of the leucaena on offer (mean 600 kg edible dry matter (EDM)/ha, 28% of total pasture EDM) was consumed. Nitrogen pools in the grass, leucaena, soil, cattle liveweight, faeces and urine were estimated. The podzolic soil (pH 4.8-5.9) was found to be deficient in P, Ca and K. Leucaena leaf tissues contained deficient levels of N, P and Ca. Grass tissues were deficient in N and P. Grazing was found to cycle 65% of N on offer in pasture herbage. However, due to the effect of the plant nutrient imbalances described above, biological N fixation by leucaena contributed only 15 kg/ha N to the pasture system over the 9-month regrowth period, of which 13 kg/ha N was cycled. Cattle retained 1.8 kg/ha N (8% of total N consumed) in body tissue and the remainder was excreted in dung and urine in approximately equal proportions. Mineral soil N concentrations did not change significantly (-3.5 kg/ha N) over the trial period. The ramifications of grazing and fertiliser management strategies, and implications for pasture rundown and sustainability are discussed.

Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM 156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Nitrification of high strength ammonia wastewtaer treatment - process selection is the major factor.

Biological nitrogen removal via the nitrite pathway in wastewater treatment is very important in Saving the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulation were carried out in a biofilm airlift reactor with autotrophic nitrifying biofilm. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.5 to 3.5 kg N/m3.d. Nitrite accumulation was stably achieved by the selective inhibition of nitrite oxidizers with free ammonia and dissolved oxygen limitation. Stable 100% conversion to nitrite could also be achieved even under the absence of free ammonia inhibition on nitrite oxidizers. Batch ammonium oxidation and nitrite oxidation with nitrite accumulating nitrifying biofilm showed that nitrite Oxidation was completely inhibited when free ammonia is higher than 0.2 mg N/L. However, nitrite oxidation activity was recovered as soon as the free ammonia concentration was below the threshold level when dissolved oxygen concentration was not the limiting factor. Fluorescence in situ hybridization analysis of cryosectioned nitrite accumulating nitrifying biofilm showed that the β-subclass of Proteobacteria, where ammonia oxidizers belong, was distributed outside the biofilm whereas the α-subclass of Proteobacteria, where nitrite oxidizers belong, was found mainly in the inner part of the biofilm. It is likely that dissolved oxygen deficiency or limitation in the inner part of the nitrifying biofilm, where nitrite oxidizers exist, is responsible for the complete shut down of the nitrite oxidizers activity under the absence of free ammonia inhibition.

Nitrate ammonification and its relationship to the accumulation of ammonium in a Vertisol subsoil

High concentrations of ammonium ( up to 270 kg N/ha) have been observed in a Vertisol soil below 1 m depth near Warra in south-east Queensland. This study examined the possibility that increased water movement into the subsoil after the removal of native vegetation, and a subsequent increase in periods of waterlogging, could have triggered nitrate ammonification and be responsible for the production of ammonium. Two incubation experiments were conducted to test this hypothesis. The first involved the incubation of repacked cores that had been amended with 30 mg N/kg of 5 atom% N-15 nitrate under low oxygen conditions for a period of 360 days. Over this time period the N-15 enrichment of the exchangeable ammonium fraction was monitored in order to detect any reduction of nitrate to ammonium. The second experiment involved the incubation of soil amended with 30 mg N/ kg of 5 atom% N-15 nitrate under waterlogged and low oxygen conditions for 75 days. During this period the redox potential of the soil was monitored using a field test to determine if reducing conditions would develop in this soil over a period of waterlogging, combined with the monitoring of any nitrate reduction to ammonium. The results of these experiments indicated that a small amount of nitrate ammonification (< 0.1 mg N/ kg) could be observed in the Warra subsoil, but that unless the rate of reduction were to significantly increase with time, this could not account for the accumulation of ammonium observed in the field. The environmental conditions that would make either dissimilatory or abiotic nitrate ammonification favourable were not observed to develop. Consequently, it has been concluded that the observed nitrate ammonification occurred via an assimilatory pathway. Due to the low rate of microbial activity in this subsoil it is considered unlikely that this process was responsible for the subsoil ammonium accumulation at Warra.

Subsoil nitrogen mineralisation and its potential to contribute to NH4 accumulation in a Vertosol

High concentrations of NH4+ (up to 270 kg N/ha) have been observed in a Vertosol below 1 m depth in south-east Queensland. This study examined the possibility that mineralisation associated with the removal of native vegetation (Acacia harpophylla) for cropping was responsible for the production of NH4+. Particularly, the potential contribution of decomposing root material and/or dissolved organic nitrogen (DON) leached into the subsoil after clearing was investigated. The amount of N that was contained within native vegetation root material was determined from an area of native vegetation adjacent to the cleared site containing elevated NH4+ concentrations. In addition, the amount of NH4+ that could be mineralised in the native vegetation soil was determined by monitoring NH4+ concentrations over 360 days in intact cores, and by conducting waterlogged incubations. To determine the rate at which a source of DON leached into the subsoil would mineralise, soil was amended with glutamic acid at a rate of 250 mg N/kg and placed under waterlogged incubation. The possibility that the acidic pH of the subsoil, or the lack of a significant subsoil microbial population, was inhibiting mineralisation was also examined by increasing soil pH from 4.4 to 7.0, and inoculating the subsoil with surface soil microorganisms during waterlogged incubations. Low concentrations of N, approximately 90 kg N/ha between 1.2 and 3 m, were found in the native vegetation root material. In addition, no net N mineralisation was observed in either the extended incubation of intact cores or in the control samples of the waterlogged incubations. Net N mineralisation was also not detected when the subsoil was amended with a source of organic N. Results indicate that this lack of mineralisation is largely due to pH inhibition of the microbial population. It is concluded that the mineralisation of either in situ organic material, or DON transported to the subsoil during leaching events, is unlikely to have significantly contributed to the subsoil NH4 accumulation at the study site.

Synthesis of optically complex core-shell colloidal suspensions: Pathways to multiplexed biological screening

The ability to generate enormous random libraries of DNA probes via split-and-mix synthesis on solid supports is an important biotechnological application of colloids that has not been fully utilized to date. To discriminate between colloid-based DNA probes each colloidal particle must be 'encoded' so it is distinguishable from all other particles. To this end, we have used novel particle synthesis strategies to produce large numbers of optically encoded particle suitable for DNA library synthesis. Multifluorescent particles with unique and reproducible optical signatures (i.e., fluorescence and light-scattering attributes) suitable for high-throughput flow cytometry have been produced. In the spectroscopic study presented here, we investigated the optical characteristics of multi-fluorescent particles that were synthesized by coating silica 'core' particles with up to six different fluorescent dye shells alternated with non-fluorescent silica 'spacer' shells. It was observed that the diameter of the particles increased by up to 20% as a result of the addition of twelve concentric shells and that there was a significant reduction in fluorescence emission intensities from inner shells as an increasing number of shells were deposited.