A study of gene expression in Legionella pneumophila biofilms through the use of confocal microscopy

Legionella pneumophila is the causative agent of Legionnaires\u27 Disease. L. pneumophila is ubiquitous in freshwater environments as well as in man-made water systems such as air conditioners and cooling towers. Legionella biofilms in these systems have been identified as the source of a number of outbreaks. Gene expression in planktonic phase L. pneumophila has been well characterized but little analysis has been conducted within biofilms. We hypothesize that gene expression in Legionella biofilms will exhibit unique expression patterns as compared to planktonic cells. To test this hypothesis Legionella were transformed with reporter gene vectors and biofilms grown on glass slides and imaged using confocal microscopy. Characterization of biofilm stages was conducted from attachment through dispersal. Gene expression of the global regulatory protein, CsrA, and the flagellar gene, FlaA, was quantified over 120hr of biofilm growth. Biofilms were imaged at five key time points in the biofilm development: 12 hr (initial attachment), 24hr (irreversible attachment), 48hr (early maturation), 72hr (late maturation), 96hr (mature biofilm) and 120hr (mature biofilm with dispersal). Whole biofilm fluorescence was measured with syto59 staining and compared to the percentage of cells that demonstrated GFP fluorescence from the reporter gene. DIC images clearly demonstrate that Legionella biofilms follow the typical biofilm developmental stages. Analysis of the CsrA expression showed upregulation in early biofilms but little to no CsrA expression in mature biofilms. FlaA was expressed in early biofilms and during late biofilms where dispersal was occurring. Planktonic cultures are often used to characterize cycles of gene expression which are often not identical to the patterns seen in biofilms. Legionella biofilms are not well characterized molecularly and here we present the first evidence showing gene expression patterns of essential genes over time within biofilms. Use of confocal microscopy for such assays provides a high resolution, specific image that allows for quantification and detailed analysis of gene expression. This research begins the opportunity to better understand biofilm gene expression that can lead to improved prevention and control of infectious biofilms

CU and the CDC

Legionella is a gram-negative genus of bacteria that is the cause of Legionnaires disease. Currently, 50 species and 70 serogroups of Legionella have been identified. The Center for Disease Control (CDC) in Atlanta maintains a bank of identified and unidentified Legionella samples. The availability of sequencing technologies has increased since many samples were collected allowing for identification of many previously unidentifiable isolates. We received 68 unidentified samples from the CDC. A sequence based typing scheme was used for characterization. Genomic DNA was extracted from samples and polymerase chain reactionwas performed on the 16S and mip genes. These samples were then sequenced at Clemson University Genomics Institute. Currently, we have identified several samples which were previously undescribed. Once a sample is identified as novel, characterization through sequencing other genes along with morphological and biochemical assays will be conducted. As a collaborative project, regular meetings occur with scientists from the CDC. Characterization of novel strains expands this lab\u27s ability to conduct outbreak analysis and risk assessment along with expanding our knowledge of the pathogen

Repeated exposure to physiologically effective doses of contraceptive hormones ethinyl estradiol and levonorgestrel do not alter the reinforcing effects of a brief visual stimulus in ovary-intact rats

Estradiol and progesterone potentiate and attenuate reward processes, respectively. Despite these well-characterized effects, there is minimal research on the effects of synthetic estrogens (e.g., ethinyl estradiol, or EE) and progestins (e.g., levonorgestrel, or LEVO) contained in clinically-utilized hormonal contraceptives. The present study characterized the separate effects of repeated exposure to EE and LEVO on responding maintained by a reinforcing visual stimulus. Forty ovary-intact female Sprague-Dawley rats received either sesame oil vehicle (n=16), 0.18 µg/day EE (n=16), or 0.6 µg/day LEVO (n=8) subcutaneous injections 30-min before daily one-hour sessions. Rats’ responding was maintained by a 30-sec visual stimulus on a Variable Ratio-3 schedule of reinforcement. The day after rats’ last session, we determined rats estrous cycle phase via vaginal cytology before sacrifice and subsequently weighing each rat’s uterus to further verify the contraceptive hormone manipulation. The visual stimulus functioned as a reinforcer, but neither EE or LEVO enhanced visual stimulus maintained responding. Estrous cytology was consistent with normal cycling in vehicle rats and halting of normal cycling in EE and LEVO rats. EE increased uterine weights consistent with typical uterotrophic effects observed with estrogens, further confirming the physiological impacts of our EE and LEVO doses. In conclusion, physiologically effective doses of EE and LEVO did not alter the reinforcing efficacy of a visual stimulus reinforcer. Future research should characterize the effects of hormonal contraceptives on responding maintained by other reinforcer types to determine the generality of the present findings

Designing an Intelligent User Interface for Preventing Phishing Attacks

Part 3: Workshop on Handling Security, Usability, User Experience and Reliability in User-Centered Development ProcessesInternational audienceMost phishing sites are simply copies of real sites with slight features distorted or in some cases masqueraded. This property of phishing sites has made them difficult for humans and various anti-phishing techniques to detect. Also, the attacker community has proved itself able to quickly adapt to anti-phishing measures, mainly warning messages to help limit the effectiveness of phishing attacks and protect unsuspecting users. Despite the notable advances made in the last years by the active warning messages for phishing, this attack remains one of the most effective. In this paper we propose an intelligent warning message mechanism, that might limit the effectiveness of phishing attacks and that might increase the user awareness about related risks. It implements an intelligent behavior that, besides warning the users that a phishing attack is occurring, explains why the specific suspect site can be fraudulent