An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

Intracellular regulation of cell signaling cascades: how location makes a difference

Organelles such as endosomes and the Golgi apparatus play a critical role in regulating signal transmission to the nucleus. Recent experiments have shown that appropriate positioning of these organelles within the intracellular space is critical for effective signal regulation. To understand the mechanism behind this observation, we consider a reaction-diffusion model of an intracellular signaling cascade and investigate the effect on the signaling of intracellular regulation in the form of a small release of phosphorylated signaling protein, kinase, and/or phosphatase. Variational analysis is applied to characterize the most effective regions for the localization of this intracellular regulation. The results demonstrate that signals reaching the nucleus are most effectively regulated by localizing the release of phosphorylated substrate protein and kinase near the nucleus. Phosphatase release, on the other hand, is nearly equally effective throughout the intracellular space. The effectiveness of the intracellular regulation is affected strongly by the characteristics of signal propagation through the cascade. For signals that are amplified as they propagate through the cascade, reactions in the upstream levels of the cascade exhibit much larger sensitivities to regulation by release of phosphorylated substrate protein and kinase than downstream reactions. On the other hand, for signals that decay through the cascade, downstream reactions exhibit larger sensitivity than upstream reactions. For regulation by phosphatase release, all reactions within the cascade show large sensitivity for amplified signals but lose this sensitivity for decaying signals. We use the analysis to develop a simple model of endosome-mediated regulation of cell signaling. The results demonstrate that signal regulation by the modeled endosome is most effective when the endosome is positioned in the vicinity of the nucleus. The present findings may explain at least in part why endosomes in many cell types localize near the nucleus.     

Neurotoxicity,drugs of abuse,and the CuZn-superoxide dismutase transgenic mice

Administration of methamphetamine (METH) to animals causes loss of DA terminals in the brain. The manner by which METH causes these changes in neurotoxicity is not known. We have tested the effects of this drug in copper/zinc (CuZn)-superoxide dismutase transgenic (SOD Tg) mice, which express the human CuZnSOD gene. In nontransgenic (non-Tg) mice, acute METH administration causes significant decreases in DA and dihydroxyphenylacetic acid (DOPAC) in the striata of non-Tg mice. In contrast, there were no significant decreases in striatal DA in the SOD Tg mice. The effects of METH on DOPAC were also attenuated in SOD Tg mice. Chronic METH administration caused decreases in striatal DA and DOPAC in the non-Tg mice, but not in the SOD-Tg mice. Similar studies were carried out with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), which also causes striatal DA and DOPAC depletion. As in the case of METH, MPTP causes marked depletion of DA and DOPAC in the non-Tg mice, but not in the SOD Tg mice. These results suggest that the mechanisms of toxicity of both METH and MPTP involve superoxide radical formation.     

NK cells: An attractive candidate for cancer therapy

Natural killer (NK) cells have significant capability in tumor immune-surveillance. The ability of lyse transformed cells immediately in an antigen-independent manner make them an attractive candidate for cancer cell therapy. Despite employment of NK cells in cancer immunotherapy, clinical trials are faced with serious limitations such as trouble with the penetration of NK cells in tumor sites, limited in vivo persistence, and tumor microenvironment interference. Taken together, the NK-cell cancer therapy is still infant scenario that has a long way to be translated in clinic. Current article first reviews characteristic features of NK lymphocytes. Then, it discusses about important disruptive barriers and motivator in the developmental stages of NK cells like as tumor microenvironment. Finally, some revolutionary approaches are highlighted utilizing of NK cells in cancer therapy.     

Identification of chalcone synthase genes and their expression patterns reveal pollen abortion in cotton

Chalcone synthase (CHS) is a key enzyme and producing flavonoid derivatives as well play a vital roles in sustaining plant growth and development. However, the systematic and comprehensive analysis of CHS genes in island cotton (G. barbadense) has not been reported yet especially response to cytoplasmic male sterility (CMS). To fill this knowledge gap, a genome-wide investigation of CHS genes were studied in island cotton. A total of 20 GbCHS genes were identified and grouped into five GbCHSs. The gene structure analysis revealed that most of GbCHS genes consisted of two exons and one intron, and 20 motifs were identified. Twenty five pairs duplicated events (12 GbCHS genes) were identified including 23 segmental duplication pairs and two tandem duplication events, representing that GbCHS gene family amplification mainly owned to segmental duplication events and evolving slowly. Gene expression analysis exhibited that the GbCHS family genes presented a diversity expression patterns in various organs of cotton. Coupled with functional predictions and gene expression, the abnormal expression of GbCHS06, 10, 16 and 19 might be associated with pollen abortion of CMS line in island cotton. Conclusively, GbCHS genes exhibited diversity and conservation in many aspects, which will help to better understand functional studies and a reference for CHS research in island cotton and other plants.