Listeria monocytogenes serotype 4b is responsible for a high percentage of fatal cases of food-borne infection. In a previous study, we created 15 monoclonal antibodies (MAbs) against a ∼77 kDa antigen that is associated with the cell surface of live L. monocytogenes serotype 4b cells. Here we report an extensive characterization of these MAbs to further their development as diagnostic reagents. The ∼77 kDa target antigen was identified by mass spectrometry and N-terminal sequencing to be IspC, a novel surface associated autolysin. Epitope localization experiments revealed that each of the 15 MAbs recognized the C-terminal cell-wall binding domain of IspC. The presence of IspC was shown to be highly conserved within L. monocytogenes serotype 4b, as evidenced by a strong reaction between anti-IspC MAbs and all 4b isolates. To determine the range of cross-reactivity with other L. monocytogenes serotypes ELISA was used to test each MAb against multiple isolates from each of the L. monocytogenes serotypes. Of the 15 MAbs, five: M2774, M2775, M2780, M2790 and M2797, showed specificity for L. monocytogenes serotype 4b and only cross reacted with serotype 4ab isolates. The kinetics of the interaction between each of the MAbs and IspC was measured using surface plasmon resonance. The MAbs M2773, M2792, M2775, M2797 and M2781 each had very low dissociation constants (4.5 × 10−9 to 1.2 × 10−8 M). While several of these antibodies have properties which could be useful in diagnostic tests, the combined high fidelity and affinity of M2775 for the IspC protein and serotype 4b isolates, makes it a particularly promising candidate for use in the development of a specific L. monocytogenes serotype 4b diagnostic test.

An optofluidic device is demonstrated with photonic components integrated onto the chip for use in fluorescence and scatter detection and counting applications. The device is fabricated by integrating the optical and fluidic components in a single functional layer. Optical excitation on-chip is accomplished via a waveguide integrated with a system of lenses that reforms the geometry of the beam in the microfluidic channel into a specific shape that is more suitable for reliable detection. Separate counting tests by detecting fluorescence and scattered signals from 2.5 and 6.0 μm beads were performed and found to show detection reliability comparable to that of conventional means of excitation and an improvement over other microchip-based designs.

Background

Drought is one of the main environmental factors limiting tree growth and productivity of plantation forests worldwide. Populus hopeiensis Hu et Chow is one of the most important commercial plantation tree species in China. However, the genes controlling drought tolerance in this species have not been identified or characterized. Here, we conducted differential expression analyses and identified a number of genes that were up- or downregulated in P. hopeiensis during water stress. To the best of our knowledge, this is the first comprehensive study of differentially expressed genes in water-stressed P. hopeiensis.

Results

Using the cDNA-AFLP detection technique, we used 256 primer combinations to identify differentially expressed genes in P. hopeiensis during water stress. In total, 415 transcript derived-fragments (TDFs) were obtained from 10× deep sequencing of 473 selected TDFs. Of the 415 TDFs, 412 were annotated by BLAST searches against various databases. The majority of these genes encoded products involved in ion transport and compartmentalization, cell division, metabolism, and protein synthesis. The TDFs were clustered into 12 groups on the basis of their expression patterns. Of the 415 reliable TDFs, the sequences of 35 were homologous to genes that play roles in short or long-term resistance to drought stress. Some genes were further selected for validation of cDNA-AFLP expression patterns using real-time PCR analyses. The results confirmed the expression patterns that were detected using the cDNA-AFLP technique.

Conclusion

The cDNA-AFLP technique is an effective and powerful tool for identifying candidate genes that are differentially expressed under water stress. We demonstrated that 415 TDFs were differentially expressed in water-stressed poplar. The products of these genes are involved in various biological processes in the drought response of poplar. The results of this study will aid in the identification of candidate genes of future experiments aimed at understanding this response of poplar.

Intracerebral hemorrhage (ICH) can cause secondary brain damage through inflammation-related pathways. Thrombin and one of its receptors, protease activated receptor-1 (PAR-1); matrix metalloproteinase (MMP)-9; and aquaporin (AQP)-4 are stroke-related inflammatory mediators that have been implicated in ICH pathology. To further characterize the inflammatory response after ICH, we studied the temporal profile of the expression of these inflammatory mediators and assessed their potential correlation with brain edema formation after brain hemorrhage in rats. ICH was modeled by infusing autologous blood into the striatum. Then mRNA and protein expression was assessed over the course of 5 days. We found that the mRNA and/or protein expression of thrombin, PAR-1, AQP-4, and MMP-9 was upregulated between 2 h and 5 days after ICH. Each reached a maximal level at day 2, except for AQP-4 protein, which peaked at day 5. Brain water content after ICH presented a similar trend; it was increased at 2 h, peaked at day 2, and then decreased but remained elevated at day 5. Our data provide novel evidence that upregulation of these selected inflammatory mediators occurs very early and persists for several days after ICH, and that temporal patterns of expression of thrombin and AQP-4 are associated with brain edema formation. These findings have important implications for efforts to reduce secondary brain damage after ICH.

This histopathologic case-control study was designed to characterize the dynamic changes in protein expression of nuclear factor-kappa B (NF-κB)/p65 subunit, macrophage inflammatory protein-2 (MIP-2), and matrix metalloproteinase-9 (MMP-9) in postmortem brains of patients with and without intracerebral hemorrhage (ICH). Thirty-six human brains from patients with ICH and six control brains were included in this study. We found that expression levels of NF-κB/p65, MIP-2, and MMP-9 were each upregulated on the injured side of the hippocampus at times ranging from 2 hr to 5 days post-ICH. Interestingly, the expression of all three markers was also upregulated on the uninjured side of the hippocampus and in the cerebellum, although to a lesser extent. These data suggest that inflammation occurs early and persists for several days after ICH in humans and could be involved in the progression of ICH-induced secondary brain damage.

Salmonella enterica subsp. enterica serotype Typhimurium is one of the major causative agents of human gastroenteritis. Here we raised a panel of 45 monoclonal antibodies (MAbs) against ser. Typhimurium DT104 by immunizing mice with formalin-killed bacteria and demonstrated that all the MAbs recognized the bacterial lipopolysaccharide (LPS) antigen. These MAbs were specific for group O:4 Salmonella with very little or no cross-reactivity with other closely related bacteria and were able to bind to the cell surface of live bacterial cells, making them potential candidates for capture and concentration of the pathogen in food and water samples. Epitope characterization revealed that the O:5 antigen present in the LPS of some serogroup 4 Salmonella is the critical factor for the binding of these MAbs to LPS. This study has provided some insights into the structure of the Salmonella LPS and its influence on the antigenicity of LPS.

An optimal excitation beam shape is necessary to perform reliable flow cytometric analysis but has so far not been implemented in a photonic-microfluidic integrated (i.e. optofluidic) device. We have achieved this feature by integrating a 1D lens system with planar waveguides and microfluidic channel on a substrate using one patterning material via a one-shot process. In this paper, we report the method of design and the performance of specifically formed excitation regions shaped to be ideal for reducing double detections, improving SNR, and for reliable detection in a flow cytometry application. Demonstration of different sizes via changes to lens design shows the ability to control the width of the shaped beam according to a targeted detection.