Antimicrobial resistance characteristics and fitness of Gram-negative fecal bacteria from volunteers treated with minocycline or amoxicillin.

A yearlong study was performed to examine the effect of antibiotic administration on the bacterial gut flora. Gram-negative facultative anaerobic bacteria were recovered from the feces of healthy adult volunteers administered amoxicillin, minocycline or placebo, and changes determined in antimicrobial resistance (AMR) gene carriage. Seventy percent of the 1039 facultative anaerobic isolates recovered were identified by MALDI-TOF as Escherichia coli. A microarray used to determine virulence and resistance gene carriage demonstrated that AMR genes were widespread in all administration groups, with the most common resistance genes being bla TEM, dfr, strB, tet(A), and tet(B). Following amoxicillin administration, an increase in the proportion of amoxicillin resistant E. coli and a three-fold increase in the levels of bla TEM gene carriage was observed, an effect not observed in the other two treatment groups. Detection of virulence genes, including stx1A, indicated not all E. coli were innocuous commensals. Approximately 150 E. coli collected from 6 participants were selected for pulse field gel electrophoresis (PFGE), and a subset used for characterisation of plasmids and Phenotypic Microarrays (PM). PFGE indicated some E. coli clones had persisted in volunteers for up to 1 year, while others were transient. Although there were no unique characteristics associated with plasmids from persistent or transient isolates, PM assays showed transient isolates had greater adaptability to a range of antiseptic biocides and tetracycline; characteristics which were lost in some, but not all persistent isolates. This study indicates healthy individuals carry bacteria harboring resistance to a variety of antibiotics and biocides in their intestinal tract. Antibiotic administration can have a temporary effect of selecting bacteria, showing co-resistance to multiple antibiotics, some of which can persist within the gut for up to 1 year

A conserved role for LRRK2 and Roco proteins in the regulation of mitochondrial activity

Parkinson’s Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo. To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects

Acridine-decorated cyclometallated gold(III) complexes: synthesis and anti-tumour investigations

(C^N) and (C^N^C) cyclometalated Au(III) represent a highly promising class of potential anticancer agents. We report here the synthesis of seven new cyclometalated Au(III) complexes with five of them bearing an acridine moiety attached via (N^O) or (N^N) chelates, acyclic amino carbenes (AAC) and N-heterocyclic carbenes (NHC). The antiproliferative properties of the different complexes were evaluated in vitro on a panel of cancer cells including leukaemia, lung and breast cancer cells. We observed a trend between the cytotoxicity and the intracellular gold uptake of some representative compounds of the series. Some of the acridine-decorated complexes were demonstrated to interact with ds-DNA using FRET-melting techniques

The Rad9.M50 Variant of the DNA Damage Checkpoint Protein Rad9 Regulates the MAP Kinase pathway in the Response to Heat Stress

Environmental stress activates the MAP kinase pathway to regulate transcription and cell division. In this pathway, a MAP kinase kinase kinases (MAPKKK) activates the MAP kinase kinase (MAPKK) which in turn phosphorylates the MAP kinase (MAPK) simultaneously at a serine or threonine residue and a tyrosine residue in close vicinity. In the fission yeast Schizosaccharomyces pombe, the dual specific MAPKK Wis1 phosphorylates the MAPK Sty1/Spc1 at threonine 171 and tyrosine 173. Active Sty1 phosphorylates and activates the transcription factor Atf1. Sty1 is closely related to Hog1 in the budding yeast S.cerevisiae and p38 in human cells. This thesis reveals a novel regulation of Sty1 by the alternative translation product of the DNA damage checkpoint protein Rad9. Alternative translation from the internal AUG codon at position 50 produces an N-terminally truncated protein variant (Rad9-M50), expression of which is restricted to dividing cells. The basal level of this variant is low and increases in the response to heat stress and DNA alkylation by the DNA damaging drug methyl-methanesulfonate (MMS). The key finding reported here is the ability of Rad9 variant to shield Sty1 from dual specific kinases other than Wis1. Loss of Wis1 eliminates the phosphorylation of Sty1 at T171 and Y173. However, removal of the Rad9 variant (rad9-M50A) restores Sty1 phosphorylation in the absence of Wis1 at high temperature and in the presence of oxidative stress. This aberrant stimulation enables Sty1 to phosphorylate Atf1. A model is presented in which Rad9 variant shields Sty1 from an alternative dual-specific kinase, possibly Wee1, Hhp1 (CK1) or Mps1/TTK. The thesis also reports the requirement of the DEAD-box RNA helicase Ded1 for the expression of Rad9 variant and presents genetic evidence linking Rad9 variant with the phosphatase Ptc1 in the response to heat stress and MMS induced DNA damage

Metasomatic events in the lithospheric mantle beneath NW Libya: petrological evidence from the Gharyan peridotite xenoliths

none6---noneBeccaluva L.; Bianchini G.; Ellam R.M.; Marzola M.; Oun K.M.; Siena F.Beccaluva, Luigi; Bianchini, Gianluca; Ellam, R. M.; Marzola, Massimiliano; Oun, K. M.; Siena, Franc

High throughput phenotypic analysis of 'Mycobacterium tuberculosis' and 'Mycobacterium bovis' strains' metabolism using Biolog Phenotype Microarrays

Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has generated interesting data worthy of further investigation