Anti-quorum sensing agents from south Florida medicinal plants and their attenuation of Pseudomonas aeruginosa pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus . Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Ethnic groups and institutions: A study of institutional engineering in four Central and Eastern European countries

The issue of institutional engineering has gained a renewed interest with the democratic transitions of the Central and Eastern European countries, as for some states it has become a matter of state survival. The four countries examined in the study – Macedonia, Slovakia, Romania and Bulgaria – exemplify the difficulty in establishing a stable democratic society in the context of the resurgence of national identity. The success of ethnonational minorities in achieving the desired policies affirming or expanding their rights as a group was conditioned upon the cohesion of the minority as well as the permissiveness of state institutions in terms of participation and representation of minority members. The Hungarian minorities in Slovakia and Romania, the Turkish minority in Bulgaria, and the Albanian minority in Macedonia, formed their political organizations to represent their interests. However, in some cases the divergence of strategies or goals between factions of the minority group seriously impeded its ability to obtain the desired concessions from the majority. The difficulty in the pursuit of policies favoring the expansion of minority rights was further exacerbated in some of the cases by the impermissiveness of political institutions. The political parties representing the interest of ethnonational minorities were allowed to participate in elections, although not without suspicions about their intent and even strong opposition from majority groups, but participation in elections and subsequent representation in legislative bodies did not translate into adoption of the desired policies. The ethnonational minorities' inability to effectively influence the decision-making process was the result of the inadequacy of democratic institutions to process these demands and channel them through the normal political process in the absence of majority desire to accommodate them. Despite the promise of democratic institutions to bring about a major overhaul of the policies of forceful assimilation and disregard for minority rights, the four cases analyzed in the study demonstrate that in effect ethnonational minorities continued to be at the mercy of the majority, especially if the minority was unable to position itself as a balancing actor.

Cancer therapy combining the modalities of hyperthermia and chemotherapy: In vitro cellular response after rapid heat accumulation in the cancer cell

Hyperthermia is usually used at a sub-lethal level in cancer treatment to potentiate the effects of chemotherapy. The purpose of this study is to investigate the role of heating rate in achieving synergistic cell killing by chemotherapy and hyperthermia. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. The cytotoxicity, mode of cell death, induction of thermal tolerance and P-gp mediated MDR following the two different modes of heating were studied. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. A slow rate hyperthermia was provided by a cell culture incubator. The results show that the potentiating effect of hyperthermia to chemotherapy can be maximized by increasing the rate of heating as evident by the results from the cytotoxicity assay. When delivered at the same thermal dose, a rapid increase in temperature from 37°C to 43°C caused more cell membrane damage than gradually heating the cells from 37°C to 43°C and thus allowed for more intracellular accumulation of the chemotherapeutic agents. Different modes of cell death are observed by the two hyperthermia delivery methods. The rapid rate laser-ICG hyperthermia @ 43°C caused cell necrosis whereas the slow rate incubator hyperthermia @ 43°C induced very mild apoptosis. At 43°C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified. This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

Role of Pseudomonas aeruginosa PA4157in Quorum Sensing and Iron Transport Regulation

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a pnmary contributing factor responsible for the morbidity and mortality in patients with cystic fibrosis. One of the trademarks of P. aeruginosa is its ability to resist antibiotics. P. aeruginosa does so in part through the LysR-type transcription factor, AmpR. To identify additional members of the AmpR regulon, a new algorithm called iterative enhancement of motifs was used to identify putative AmpR binding sites upstream of open reading frames in the P. aeruginosa genome. The surprising primary hit of this analysis was the promoter of an uncharacterized open reading frame, P A 415 7. P A 415 7 is located upstream ofthefep operon, which is known to be involved in iron acquisition. PA4157 shares high homology to the IclR family of transcriptional regulators which are known to regulate quorum sensing (QS), an elaborate cell-cell communication signaling system that uses quoromones. We postulated two hypotheses: 1) AmpR regulation of QS genes is mediated by PA4157, and 2) PA4157 may be involved in iron acquisition. To address the role of P A 415 7 we generated an in-frame chromosomal deletion of P A 415 7 in P. aeruginosa PA01 (PA0 PA4157). We compared PA0 PA4157 with its parent strain P A0 1 for its ability to produce quoromones using Chromobacterium violaceum as an indicator strain and LasA proteases using Staphylococcus aureus. We also tested its role in virulence using a Caenorhabditis elegans killing assay. Growth in iron-deficient media was also examined to determine if P A4157 has a potential role in iron uptake regulation. Our preliminary results suggest that P A 415 7 is not involved in quorum sensing regulation but does seem to exert a negative regulatory effect on iron uptake in P. aeruginosa P A0 1.

"Unfair'' Restaurant Reviews:To Sue Or Not To Sue

In their discussion entitled - “Unfair” Restaurant Reviews: To Sue Or Not To Sue - by John Schroeder and Bruce Lazarus, Assistant Professors, Department of Restaurant, Hotel and Institutional Management at Purdue University, the authors initially state: “Both advantages and disadvantages exist on bringing lawsuits against restaurant critics who write “unfair” reviews. The authors, both of whom have experience with restaurant criticism, offer practical advice on what realistically can be done by the restaurateur outside of the courtroom to combat unfair criticism.” Well, this is going to be a sticky wicket no matter how you try to defend it, reviews being what they are; very subjective pieces of opinionated journalism, especially in the food industry. And, of course, unless you can prove malicious intent there really is no a basis for a libel suit. So, a restaurateur is at the mercy of written opinion and the press. “Libel is the written or published form of slander which is the statement of false remarks that may damage the reputation of others. It also includes any false and malicious publication which may damage a person's business, trade, or employment,” is the defined form of the law provided by the authors. Anecdotally, Schroeder and Lazarus offer a few of the more scathing pieces reviewers have written about particular eating establishments. And, yes, they can be a bit comical, unless you are the owner of an establishment that appears in the crosshairs of such a reviewer. A bad review can kneecap even a popular eatery. “Because of the large readership of restaurant reviews in the publication (consumer dining out habits indicate that nearly 50 percent of consumers read a review before visiting a new restaurant) your business begins a very dangerous downward tailspin,” the authors reveal, with attribution. “Many restaurant operators contend that a bad review can cost them an immediate trade loss of upward of 50 percent,” Schroeder and Lazarus warn. “The United States Supreme Court has ruled that a restaurant owner can collect damages only if he proves that the statement or statements were made with “actual malice,” even if the statements were untrue,” the authors say by way of citation. And that last portion of the statement cannot be over-emphasized. The first amendment to the U.S. Constitution does wield a heavy hammer, indeed, and it should. So, what recourse does a restaurateur have? The authors cautiously give a guarded thumbs-up to a lawsuit, but you better be prepared to prove a misstatement of fact, as opposed to the distinguishable press protected right of opinion. For the restaurateur the pitfalls are many, the rewards few and far between, Schroeder and Lazarus will have you know. “…after weighing the advantages and disadvantages of a lawsuit against a critic...the disadvantages are overwhelming,” the authors say. “Chicago restaurant critic James Ward said that someone dumped a load of manure on his yard accompanied by a note that read - Stop writing that s--t! - after he wrote a review of a local restaurant.” Such is a novel if not legally measurable tack against an un-mutual review.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

The Sesquiterpene Lactone Dehydroleucodine Triggers Senescence and Apoptosis in Association with Accumulation of DNA Damage Markers

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.

Mapping and Assessing Fire Damage on Broadleaved Forest Communities in Big Cypress National Preserve

Within Big Cypress National Preserve (BICY), oak-dominated forests and woodlands as well as tropical and temperate hardwood hammocks are integral components of the landscape and are biodiversity hotpots for both flora and fauna. These broadleaved forest communities serve as refugia for many of the Preserve’s wildlife species during prolonged flooding and fires. However, both prolonged flooding and severe fires, which are important and necessary disturbance vectors within this landscape, can have deleterious effects on these forested communities. This is particularly true in the case of fires, which under extreme conditions associated with drought and elevated fuel loads, can burn through these forested communities consuming litter and understory vegetation and top killing most, if not all, of the trees present.

Stray dogs in Bucharest, and the political economy of Romanian socialism. From hunting the “enemies” of Stalinist economy, to poisoning urban parasites, and the rise of urban anarchy: 1950s-1990s.

My paper discusses three different ways in which stray dogs have been intertwined with ideologies of economic and urban development in Romania. I categorize results from archival and ethnographic research under three major time periods: early socialism, late socialism, and post-socialism. During early socialism stray dogs were seen to be damaging the soviet economy by killing species that humans could also hunt, like rabbits. During late socialism, stray dogs appeared as the enemies of the communist city, and the department of urban sanitation was given orders to poison dogs with strychnine. Finally, the increasing number of stray dogs in Bucharest after the collapse of communism was seen as a direct result of former communist demolitions, and was also taken as a sign of the collapsing state. Through such examples my paper discusses how the state and particular population groups have seen dogs as parts of an unwanted and dangerous nature, rather than a species that needs to be protected. I argue that distinctions of nature and culture have served discourses of civilization and the view of Bucharest as a model socialist, and then European city. Throughout my paper I juxtapose the treatment of stray dogs with other, more “valued” urban natures like the protection of parks, the wide-spread hobby of pigeon breeding during socialist years, the most recent debate on saving the rural area of Rosia Montana from non-environmentally friendly methods of gold extraction, and the current trend of healthy eating and living.

Anti-Quorum Sensing Agents from South Florida Medicinal Plants and their Attenuation of Pseudomonas Aeruginosa Pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.