Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water

Aeromonas punctata is the causative agent of septicemia, diarrhea, wound infections, meningitis, peritonitis, and infections of the joints, bones and eyes. Bacteriophages are often considered alternative agents for controlling bacterial infection and contamination. In this study, we described the isolation and preliminary characterization of bacteriophage IHQ1 (family Myoviridae) active against the Gram-negative bacterial strain A. punctata. This virulent bacteriophage was isolated from stream water sample. Genome analysis indicated that phage IHQ1 was a double-stranded DNA virus with an approximate genome size of 25–28 kb. The initial characterization of this newly isolated phage showed that it has a narrow host range and infects only A. punctata as it failed to infect seven other clinically isolated pathogenic strains, i.e., methicillin-resistant Staphylococcus aureus 6403, MRSA 17644, Acinetobacter 33408, Acinetobacter 1172, Pseudomonas aeruginosa 22250, P. aeruginosa 11219, and Escherichia coli. Proteomic pattern of phage IHQ1, generated by SDS-PAGE using purified phage particles, showed three major and three minor protein bands with molecular weights ranging from 25 to 70 kDa. The adsorption rate of phage IHQ1 to the host bacterium was also determined, which was significantly enhanced by the addition of 10 mM CaCl₂. From the single-step growth experiment, it was inferred that the latent time period of phage IHQ1 was 24 min and a burst size of 626 phages per cell. Moreover, the pH and thermal stability of phage IHQ1 were also investigated. The maximum stability of the phage was observed at optimal pH 7.0, and it was totally unstable at extreme acidic pH 3; however, it was comparatively stable at alkaline pH 11.0. At 37°C the phage showed maximum number of plaques, and the viability was almost 100%. The existence of Aeromonas bacteriophage is very promising for the eradication of this opportunistic pathogen and also for future applications such as the design of new detection and phage typing (diagnosis) methods. The specificity of the bacteriophage for A. punctata makes it an attractive candidate for phage therapy of A. punctata infections.     

Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within activin receptor 2 (ACVR2)

We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.     

Morphological Changes in the Midgut of Aedes aegypti L. (Diptera: Culicidae) Larvae Following Exposure to an Annona coriacea (Magnoliales: Annonaceae) Extract

Bioinsecticides are important in the control of disease vectors, but data regarding their physiological effects on target insects are incomplete. This study describes morphological changes that occur in the midgut of third instar Aedes aegypti L. (Diptera: Culicidae) following treatment with a methanolic extract of Annona coriacea (Magnoliales: Annonaceae). Dissected midguts were subdivided into anterior and posterior regions and analyzed by light and scanning electron microscopy. Insects exposed to the extract displayed intense, destructive cytoplasmic vacuolization in columnar and regenerative midgut cells. The apical surfaces of columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes and/or apoptosis. We report that A. coriacea extracts induced morphological alterations in the midgut of A. aegypti midgut larvae, supporting the use of plant extracts for control of the dengue vector.     

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.     

Gene expression profiling through microarray analysis in Arabidopsis thaliana colonized by Pseudomonas putida MTCC5279, a plant growth promoting rhizobacterium

Plant growth promotion is a multigenic process under the influence of many factors; therefore an understanding of these processes and the functions regulated may have profound implications. Present study reports microarray analysis of Arabidopsis thaliana plants inoculated with Pseudomonas putida MTCC5279 (MTCC5279) which resulted in significant increase in growth traits as compared with non-inoculated control. The gene expression changes, represented by oligonucleotide array (24652 genes) have been studied to gain insight into MTCC5279 assisted plant growth promotion in Arabidopsis thaliana. MTCC5279 induced upregulated Arabidopsis thaliana genes were found to be involved in maintenance of genome integrity (At5g20850), growth hormone (At3g23890 and At4g36110), amino acid synthesis (At5g63890), abcissic acid (ABA) signaling and ethylene suppression (At2g29090, At5g17850), Ca⁺² dependent signaling (At3g57530) and induction of induced systemic resistance (At2g46370, At2g44840). The genes At3g32920 and At2g15890 which are suggested to act early in petal, stamen and embryonic development are among the downregulated genes. We report for the first time MTCC5279 assisted repression of At3g32920, a putative DNA repair protein involved in recombination and DNA strand transfer in a process of rapid meiotic and mitotic division.     

On the use of infinite elements for the determination of optimal closure patterns based on stress analysis

Solid infinite elements are used in conjunction with finite elements to compute the stress and displacement distribution resulting from the suturing of wounds of symmetric and nonsymmetric shapes in orthotropic, abdominal human skin. The optimal pattern of suturing of wounds are investigated from a stress perspective. Highly accurate, quantitative and qualitative improvements over the use of finite elements to approximate distant boundaries are obtained. Numerical results quantitatively agree with analytic results computed using complex analysis techniques. The technique used and the results obtained will aid surgeons in closing nonsymmetrical wounds on regions of the body that exhibit orthotropy.     

The glutamine commute: take the N line and transfer to the A

The transfer of glutamine between cells contributes to signaling as well as to metabolism. The recent identification and characterization of the system N and A family of transporters has begun to suggest mechanisms for the directional transfer of glutamine, and should provide ways to test its physiological significance in diverse processes from nitrogen to neurotransmitter release.     

Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T-cells

In this report, we explore the mechanisms of targeting of p300 to the interleukin-2 (IL-2) promoter in response to mitogenic and oncogenic molecular signals. Recruitment of p300 by cAMP-responsive element-binding protein-Rel cross-talk at the composite CD28 response element (CD28RE)-TRE element of the IL-2 promoter is essential for promoter inducibility during T-cell activation, and CD28RE-TRE is the exclusive target of the human T-cell lymphotropic virus type I oncoprotein Tax. The intrinsic histone acetyltransferase activity of p300 is dispensable for activation of the IL-2 promoter, and the N-terminal 743 residues contain the minimal structural requirements for synergistic transactivation of the CD28RE-TRE, the IL-2 promoter, and endogenous IL-2 gene expression. Mutational analysis of p300 reveals differential structural requirements for the N-terminal p300 module by individual cis-elements within the IL-2 promoter. These findings provide evidence that p300 assembles at the IL-2 promoter to form an enhanceosome-like signal transduction target that is centrally integrated at the CD28RE-TRE element of the IL-2 promoter through specific protein module-targeted associations in activated T-cells.