Integrated response of the upper and lower respiratory tract of asthmatic subjects to frigid air.

To evaluate the influence of cold air hyperpnea on integrated upper and lower airway behavior, 22 asthmatic volunteers hyperventilated through their mouths (OHV) and noses (NHV) while pulmonary and nasal function were determined individually and in combination. In the isolated studies, OHV at a minute ventilation of 65 +/- 3 l/min lowered the 1-s forced expiratory volume (FEV(1)) 24 +/- 2% (P < 0. 001) and NHV (40 l/min) induced a 31 +/- 9% (P < 0.001) increase in nasal resistance (NR). In the combined studies, oral hyperpnea reduced the FEV(1) (DeltaFEV(1) 26 +/- 2%, P < 0.001) and evoked a significant rise in NR (DeltaNR 26 +/- 9%, P = 0.01). In contrast, NHV only affected the upper airway. NR rose 33 +/- 9% (P = 0.01), but airway caliber did not change (DeltaFEV(1) 2%, P = 0.27). The results of this investigation demonstrate that increasing the transfer of heat and water in the lower respiratory tract alters bronchial and nasal function in a linked fashion. Forcing the nose to augment its heat-exchanging activity, however, reduces nasal caliber but has no effect on the intrathoracic airways.     

Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA∝barley genomic DNA junctions

Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with thehpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a non-destructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants.     

Avian influenza (H5N1) virus of clade 2.3.2 in domestic poultry in India

South Asia has experienced regular outbreaks of H5N1 avian influenza virus since its first detection in India and Pakistan in February, 2006. Till 2009, the outbreaks in this region were due to clade 2.2 H5N1 virus. In 2010, Nepal reported the first outbreak of clade 2.3.2 virus in South Asia. In February 2011, two outbreaks of H5N1 virus were reported in the State of Tripura in India. The antigenic and genetic analyses of seven H5N1 viruses isolated during these outbreaks were carried out. Antigenic analysis confirmed 64 to 256-fold reduction in cross reactivity compared with clade 2.2 viruses. The intravenous pathogenicity index of the isolates ranged from 2.80-2.95 indicating high pathogenicity to chickens. Sequencing of all the eight gene-segments of seven H5N1 viruses isolated in these outbreaks was carried out. The predicted amino acid sequence analysis revealed high pathogenicity to chickens and susceptibility to the antivirals, amantadine and oseltamivir. Phylogenetic analyses indicated that these viruses belong to clade 2.3.2.1 and were distinct to the clade 2.3.2.1 viruses isolated in Nepal. Identification of new clade 2.3.2 H5N1 viruses in South Asia is reminiscent of the introduction of clade 2.2 viruses in this region in 2006/7. It is now important to monitor whether the clade 2.3.2.1 is replacing clade 2.2 in this region or co-circulating with it. Continued co-circulation of various subclades of the H5N1 virus which are more adapted to land based poultry in a highly populated region such as South Asia increases the risk of evolution of pandemic H5N1 strains.     

Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome

India is endemic for foot-and-mouth disease and it continues to be a major threat to the livestock industry despite vaccination programmes. In the present study, the ability of specific small interfering (si)RNAs directed against different genomic regions of foot-and-mouth disease virus (FMDV) to inhibit virus replication in BHK-21 cells was examined. For preliminary evaluation of possible siRNA-mediated FMDV inhibition, a cocktail of several unique populations of 12-30bp siRNAs were successfully produced corresponding to three target regions located at structural (VP3-VP1), non-structural (2A-2C), and non-structural-untranslated (3D-3'UTR) region of serotype Asia1. Once the populations of siRNAs generated were found to reduce the virus titre significantly, two highly conserved 21bp siRNA duplexes were designed by analysing all FMDV sequence entries available in public-domain databases. In virus titration assay, more than 99% inhibition of virus yield for all the four serotypes (type Asia1, O, A, and C) could be demonstrated in cells transfected with each of the FMDV-specific siRNAs at 24h post-infection, compared to control cells transfected with scrambled siRNA. This was well supported by reduction in OD values in FMDV-specific sandwich ELISA. Although 100-fold reduction in virus titre with siRNA1 is substantial considering the transfection efficiency and fixed level of input siRNA, siRNA2 emerged to be a better choice as target where more than 300-fold reduction was observed and its inhibitory effect extended up to 48 h post-infection against all the serotypes. Interestingly, in the present study type A virus (IND 17/77) had a single mismatch at position 2 in the siRNA2 target region but it did not abrogate the inhibitory effect.     

An improved hypergeometric probability method for identification of functionally linked proteins using phylogenetic profiles

Predicting functions of proteins and alternatively spliced isoforms encoded in a genome is one of the important applications of bioinformatics in the post-genome era. Due to the practical limitation of experimental characterization of all proteins encoded in a genome using biochemical studies, bioinformatics methods provide powerful tools for function annotation and prediction. These methods also help minimize the growing sequence-to-function gap. Phylogenetic profiling is a bioinformatics approach to identify the influence of a trait across species and can be employed to infer the evolutionary history of proteins encoded in genomes. Here we propose an improved phylogenetic profile-based method which considers the co-evolution of the reference genome to derive the basic similarity measure, the background phylogeny of target genomes for profile generation and assigning weights to target genomes. The ordering of genomes and the runs of consecutive matches between the proteins were used to define phylogenetic relationships in the approach. We used Escherichia coli K12 genome as the reference genome and its 4195 proteins were used in the current analysis. We compared our approach with two existing methods and our initial results show that the predictions have outperformed two of the existing approaches. In addition, we have validated our method using a targeted protein-protein interaction network derived from protein-protein interaction database STRING. Our preliminary results indicates that improvement in function prediction can be attained by using coevolution-based similarity measures and the runs on to the same scale instead of computing them in different scales. Our method can be applied at the whole-genome level for annotating hypothetical proteins from prokaryotic genomes.