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Mice that are incapable of expressing the small G protein Rhes have been generated and have shown to have abnormalities in behaviors mediated by the striatum, a region in which Rhes is highly expressed. Moreover, conditions that result in dopamine supersensitivity and a breakdown in D1/D2 synergism in rodents, consistently decrease rhes mRNA in striatum. Thus, Rhes may play have relevance in dopamine signal modulation. For evaluating the role of Rhes in anxiety, stereotypy and basal motor activity, adult male and female wild-type (WT) mice, Rhes knockout (KO) mice, and mice heterozygous for the KO and WT alleles (Het) were tested. There was no genotype differences in the distance traveled in the open field. However, female KO mice showed lower anxiety than either WTs or Hets, based on the quantity of time spent in the periphery vs. the central area of the open field (p<0.05). With respect to striatally-mediated motor stereotypy, the mixed D1/D2 agonist apomorphine elicited a significant greater response in male KO and Het compared to WTs (p<0.05). In previous studies of D1/D2 synergism, it has been consistently found in rats and mice that when D2 receptors alone are stimulated, there is an early and brief, D1 independent peak in stereotypy that disappears by 20 minutes. In the present study, this effect was more intense in male KO mice compared to the other two genotypes during the interval between 5 and 10 minutes (p<0.05). The current findings favor the hypothesis that the GTP-binding protein Rhes interacts with as yet unidentified cellular proteins to buffer the transduction of synaptic dopamine signals into intracellular responses. Decreased or loss of Rhes therefore results in increased DA signal transduction.

Les réponses adaptatives fonctionnelles (secondaires à des modulations de la signalisation cellulaire) peuvent contribuer à la résistance des tumeurs humaines aux traitements ciblés. Ce travail vise à caractériser les changements induits par la chimiothérapie dans le phosphokinome de cellules de cancer du côlon, et à sélectionner des combinaisons de médicaments synergiques en ciblant les kinases dont l’activation est la plus importante. Nous avons créé des scripts informatiques pour l’analyse sur le logiciel de calcul R de données provenant de tests cytotoxiques avec des matrices de combinaison de doses à deux ou trois médicaments. Nous avons profilé les changements induits par différents médicaments sur des cellules de la lignée HT29 xénogreffées dans des souris immunodéprimées. Nous avons testé le 5FU, l’oxaliplatine, l’irinotecan, le cetuximab ainsi que les combinaisons de ces médicaments. Nous avons sélectionné les kinases activées par le traitement par irinotecan, notamment AKT et MEK1, et nous avons testés in vitro et in vivo des combinaisons à deux médicaments d’inhibiteurs de ces deux kinases et l’irinotecan sur 6 lignées de cancer du côlon. Enfin nous avons testés in vitro la combinaison des trois médicaments. Nous avons observé que la combinaison d’un inhibiteurs d’AKT et de MEK ainsi que la combinaison des trois médicaments étaient caractérisées par un synergisme significatif Cette étude a démontré que la chimiothérapie induit une reprogrammation des voies de signalisation intracellulaires, et fournit le rationnel pour une évaluation du profilage de la reprogrammation du phosphokinome comme outil pour développer de nouvelles combinaisons de médicaments. / Functional (i.e. due to cellular machinery modulations) adaptive responses could also contribute to human tumor resistance to targeted drugs. We hypothesized that the activation of tumor cell kinases in response to chemotherapy treatment could render the cell depending on them, and that the inhibition of these activated kinases could achieve a synergistic effect with chemotherapy agents. We compiled R scripts for analysis of data from cytotoxic tests with dose matrix combinations of 2 or 3 drugs. We evaluated phosphokinome rewiring induced by 5FU, irinotecan, oxaliplatin and cetuximab when these drugs were used alone or in combination on mice xenografted with HT29 cell line. We observed an activation of AKT and MEK1 after irinotecan treatment, and we tested two- and three drug combinations of BKM120, an AKT inhibitor, MEK162, a MEK inhibitor, and irinotecan. We showed that BKM120 and MEK162 are synergistic, as well as the combination of the three drugs. Our study shows that chemotherapy induces a significant rewiring of intracellular signaling pathways, and that profiling phosphokinome remodeling after chemotherapy treatment is useful in order to design synergistic drug combinations.

Drug-resistant bacteria, especially those belonging to the Enterobacteriaceae family, have become increasingly problematic in the nosocomial setting. However, a solution may be to exploit bacteria’s ability to produce inhibitory proteins, like bacteriocins, to suppress competitors and synergistically pair these proteins with antibiotics. Our lab has discovered a potentially novel plasmid-mediated antimicrobial protein produced by as specific strain of Klebsiella pneumoniae. To verify the genetic elements of this plasmid necessary to produce the antimicrobial, a gene interruption plasmid library was generated by transposon mutagenesis using the EZ-TN5TM system. These transposon plasmids were then electroporated into competent E. coli. The resulting E. coli were then plated and screened on agar containing kanamycin to ensure successful plasmid uptake and were now able to secrete the antimicrobial protein. The transposon’s unique sequence allowed primer- binding sites, which were used to sequence the plasmid. Four different sequences were analyzed by NCBI BLAST comparisons and matched with high similarity to: 1) a predicted colicin; 2) an uncharacterized Klebsiella protein, 3) a TraM recognition domain containing protein. The K. pneumoniae antimicrobial protein has been shown, when spotted on lawns of Citrobacter freundii, Enterobacter cloacae and Enterobacter aerogenes to inhibit their growth. It has been additionally shown to inhibit the growth of closely related strains including Klebsiella pnuemoniae strain 9997 when spotted on a lawn. When the protein was synergistically paired with subinhibitory levels of common antibiotics, there was an increase in the effectiveness of the antibiotic, it was paired with. The optical density, MTT, and CFUs demonstrate that when the K. pneumoniae protein is paired with Streptomycin or Kanamycin, growth is inhibited greater than the antibiotic alone. These results demonstrate the importance of studying polymicrobial interactions as a means to combat drug resistance and discover novel antimicrobial derived proteins for new therapeutics.

Cancer is a group of diseases that are the second leading cause of human mortality in the United States. Discovering new therapies is vital to conquer cancer. Thymoquinone (TQ) is found in the plant Nigella sativa. TQ was found to be cytotoxic to the human ovarian cancer cell lines PA-1, CAOV-3 and SKOV-3, which have varying p53 status. PA-1 cells were the most sensitive, indicating that TQ was effective against cells having wild-type (WT) p53. Western blots indicated an increase in p53 in cell lines having WT p53. TQ when given concurrently with cisplatin resulted in antagonism for PA-1, A172 and H460 cell lines. Sequential exposure to TQ followed by cisplatin resulted in synergy or additive effects in these cell lines. Sequential exposure to cisplatin followed by TQ resulted in additive or moderate antagonism in these cell lines. Concurrent exposure to TQ and paclitaxel showed synergy in PA-1 and H460 cells. Sequential exposure to TQ followed by paclitaxel resulted in synergism or antagonism in A172, PA-1, and H460 cells. Paclitaxel followed by TQ resulted in antagonism or synergism in these cells. These results demonstrate that TQ has a potential as an antineoplastic agent and may affect p53 levels.

Exploiting the chemical ecology of an insect can unveil novel strategies for its pest management. Though much has been learned about the chemical ecology of Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), a major pest of solanaceous crops in the U.S., there has been little use of this knowledge in pest management. To better understand host plant selection by CPB, field and laboratory-choice experiments were conducted in Virginia. In laboratory studies, CPB preferred potato over both tomato and eggplant foliage and eggplant over tomato foliage. However, field studies using counts of live beetles on untreated paired plants and counts of dead beetles on insecticide-treated plants revealed no significant preference for potato over eggplant. Additional studies showed that the presence of adult male CPB on foliage greatly impacted host plant selection, with significantly more adults being attracted to eggplant with male beetles than any other treatment combination. Adult CPB have been shown to be attracted to (S)-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol [(S)-CPB I], a male-produced aggregation pheromone. Field studies were conducted to determine if the opposite enantiomer of the pheromone, (R)-CPB I had an effect on CPB in the field. Results revealed no differences in counts of all CPB life stages between untreated potato plots with and without rows inundated with (R)-CPB I lures. In addition, the relative attraction of CPB adults to various racemic forms of the (S)- and (R)-enantiomers was also investigated and showed that racemic blends that were less than 97%(S) were not attractive to CPB adults. Combinations of the (S)-CPB I pheromone with synthetic plant volatiles consisting of (Z)-3-hexenyl acetate, (+)-linalool, and methyl salicylate were investigated in a trap crop strategy in potatoes, but failed to reduce CPB numbers in untreated middle rows of potatoes. Combinations of the (S)-CPB I pheromone with synthetic plant volatiles were also used in a novel CPB trap designed to catch colonizing adults in the field. Although the traps caught CPB adults, no differences were observed in traps baited with and without the attractant. Metaflumizone, a novel semicarbazone insecticide, was recently shown to be highly efficacious on CPB. Laboratory studies found the combination of metaflumizone and a low concentration (0.39 ppm) of the pyrethroid esfenvalerate was slightly synergistic on CPB adults and early (1st-2nd) instar larvae. Field trials combining a low rate of esfenvalerate and metaflumizone at one tenth the field rate controlled beetles as well as the full rate of metaflumizone. / Ph. D.

Objectives: Aggregatibacter actinomycetemcomitans is major putative bacterial pathogen in human periodontitis, particularly in aggressive periodontitis in younger-aged individuals. Systemic administration of certain antibiotics in combination have been demonstrated to exert synergistic antimicrobial effects against A. actinomycetemcomitans, and to markedly enhance elimination or suppression of A. actinomycetemcomitans from the subgingival dental plaque microbiome of periodontitis patients beyond that attained by periodontal mechanical debridement/surgery. However, studies on antibiotic synergisms against periodontal A. actinomycetemcomitans were conducted over 20 years ago, and only involved subgingival clinical isolates of the organism from periodontitis patients in Europe. Since temporal and geographic changes in antimicrobial susceptibility are documented among periodontitis-associated microorganisms, it is not known whether or not antibiotic synergisms against periodontal A. actinomycetemcomitans are present today among clinical isolates of the organism as recovered from periodontitis patients in the United States. As a result, the purpose of the present study was to assess the potential in vitro antimicrobial synergisms between amoxicillin plus metronidazole, between ciprofloxacin plus metronidazole, and between spiramycin and metronidazole, against clinical periodontal isolates of A. actinomycetemcomitans of United States origin. Methods: Standardized cell suspensions, equivalent to a 1.0 McFarland turbidity standard, were prepared with four fresh clinical isolates of A. actinomycetemcomitans, each recovered from the subgingival microbiota of United States periodontitis subjects, and plated onto to the surfaces of 150-mm diameter culture plates containing Haemophilus test medium. After drying, antibiotic-impregnated, quantitative, gradient diffusion strips (MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, ciprofloxacin, spiramycin, and metronidazole were placed apart from each other onto the inoculated Haemophilus test medium surfaces, so that two test antibiotics per plate were employed against each A. actinomycetemcomitans clinical isolate for antibiotic susceptibility testing of individual antibiotic drugs. After 48 hours incubation in air + 5% CO2, individual MIC values for each antibiotic against A. actinomycetemcomitans were read in ug/ml at the point where the edge of the bacterial inhibition ellipse intersected with the MIC Test Strip, with the occurrence of antibiotic resistance among the A. actinomycetemcomitans clinical isolates determined using Haemophilus species antibiotic resistance breakpoint concentrations established by the United States Clinical Laboratory Standards Institute (CLSI) and the Société Francaise de Microbiologie. In vitro synergy testing of amoxicillin plus metronidazole, ciprofloxacin plus metronidazole, and spiramycin plus metronidazole was performed after determination of individual antibiotic MIC testing by placing MIC Test Strips for each of the two test antibiotics per combination in a cross formation onto the surfaces of A. actinomycetemcomitans-inculated Haemophilus test medium agar so that there was a 90° angle between the two antibiotic strips at the point where their individual MIC values against A. actinomycetemcomitans intersected on their respective MIC interpretive scales. After 48 hours incubation in air + 5% CO2, the MIC values for each antibiotic in combination against the A. actinomycetemcomitans clinical isolates was read in ug/ml where the edge of the bacterial inhibition ellipses intersected with each of the MIC Test Strips. For each of the three antibiotic combinations tested in vitro against the four A. actinomycetemcomitans clinical isolates, the fractional inhibitory concentration (FIC) index was calculated per each antibiotic in combination. FIC index values of less than or equal to 0.5 indicated the presence of synergistic antimicrobial effects of the antibiotic combination against the test bacterial clinical isolates, whereas FIC index values of greater than 0.5, but less than or equal to 4.0 indicated indifference, and FIC values greater than 4.0 represented the presence of in vitro antimicrobial antagonism between the two antibiotics in combination. Results: All of the four A. actinomycetemcomitans clinical isolates were susceptible in vitro to amoxicillin alone (all MIC values actinomycetemcomitans clinical isolates were resistant in vitro to metronidazole alone, with MIC values of resistant strains ranging between 24.0-48.0 ug/ml (resistance breakpoint threshold MIC value >16 ug/ml), and all were resistant in vitro to spiramycin alone, with MIC values of > 32.0 ug/ml exhibited by each of the four tested periodontal A. actinomycetemcomitans strains. In synergy testing, markedly lower MIC values were found for amoxicillin and metronidazole, as well as with ciprofloxacin and metronidazole, when tested in combination together as compared to being tested alone. FIC index values for the combination of amoxicillin plus metronidazole were all actinomycetemcomitans clinical isolates tested. Similarly, FIC index values for the combination of ciprofloxacin plus metronidazole were all actinomycetemcomitans clinical isolates tested. FIC index values for the combination of spiramycin plus metronidazole were > 0.5 for three of the periodontal A. actinomycetemcomitans strains, ranging from 0.875-2.0, which is indicative of an indifferent in vitro antimicrobial interaction between spiramycin and metronidazole against the three periodontal A. actinomycetemcomitans clinical isolates. The FIC index value of the combination of spiramycin plus metronidazole against a single periodontal A. actinomycetemcomitans clinical isolate was actinomycetemcomitans clinical isolate. Conclusions: Amoxicillin and ciprofloxacin individually were active against all A. actinomycetemcomitans clinical periodontal isolates, whereas most or all strains were resistant to metronidazole and spiramycin by themselves. Antimicrobial synergism was found for the combinations of amoxicillin plus metronidazole, and for ciprofloxacin plus metronidazole, against all four periodontal A. actinomycetemcomitans clinical isolates of United States origin. Spiramycin plus metronidazole generally failed to exhibit antimicrobial synergism against periodontal A. actinomycetemcomitans. These findings confirm and extend European synergism studies conducted in mid-1990s on antibiotic synergisms against periodontal A. actinomycetemcomitans, and are the first to demonstrate antibiotic synergism against United States periodontal A. actinomycetemcomitans clinical isolates. Additional research studies are needed to determine whether these in vitro synergistic effects of amoxicillin plus metronidazole, and of ciprofloxacin plus metronidazole, also occur in vivo and significantly enhance subgingival elimination or suppression of subgingival A. actinomycetemcomitans. / Oral Biology

The oral route of administration remains the preferred route of administrating drugs due to patient acceptance and compliance. Therapeutic proteins are currently mainly administered by means of the parenteral route because of its low intestinal epithelial permeation capability. The major challenges for oral delivery of proteins and peptides are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. The latter can be overcome by including safe and effective absorption enhancers in dosage forms. Aloe vera, Aloe ferox and Aloe marlothii gel materials as well as N-trimethyl chitosan chloride (TMC) were shown to be capable of increasing peptide drug transport across in vitro models such as Caco-2 cell monolayers. The purpose of this study is to investigate binary combinations of chemical drug absorption enhancers and to determine if synergistic drug absorption enhancement effects exist. A. vera, A. ferox and A. marlothii leaf gel materials as well as with N-trimethyl chitosan chloride (TMC) were combined in different ratios and their effects on the transepithelial electrical resistance (TEER) as well as the transport of FITC-dextran across Caco-2 cell monolayers were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers such as complex formation. In terms of FITC-dextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption enhancers at lower concentrations compared to single components. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

The oral route of administration remains the preferred route of administrating drugs due to patient acceptance and compliance. Therapeutic proteins are currently mainly administered by means of the parenteral route because of its low intestinal epithelial permeation capability. The major challenges for oral delivery of proteins and peptides are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. The latter can be overcome by including safe and effective absorption enhancers in dosage forms. Aloe vera, Aloe ferox and Aloe marlothii gel materials as well as N-trimethyl chitosan chloride (TMC) were shown to be capable of increasing peptide drug transport across in vitro models such as Caco-2 cell monolayers. The purpose of this study is to investigate binary combinations of chemical drug absorption enhancers and to determine if synergistic drug absorption enhancement effects exist. A. vera, A. ferox and A. marlothii leaf gel materials as well as with N-trimethyl chitosan chloride (TMC) were combined in different ratios and their effects on the transepithelial electrical resistance (TEER) as well as the transport of FITC-dextran across Caco-2 cell monolayers were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers such as complex formation. In terms of FITC-dextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption enhancers at lower concentrations compared to single components. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015