Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.     

Synthesis and evaluation of alkenyl indazoles as selective Aurora kinase inhibitors

A series of alkenyl indazoles were synthesized and evaluated in Aurora kinase enzyme assays. Several promising leads were optimized for selectivity towards Aurora B. Excellent binding affinity and good selectivity were achieved with optimized compounds in isolated Aurora subfamily assays.     

A potential tocopherol acetate loaded palm oil esters-in-water nanoemulsions for nanocosmeceuticals

Background  

Cosmeceuticals are cosmetic-pharmaceutical hybrids intended to enhance health and beauty of the skin. Nanocosmeceuticals use nano-sized system for the delivery of active ingredients to the targeted cells for better penetration. In this work, nanoemulsion from palm oil esters was developed as a delivery system to produce nanocosmeceuticals. The stability of the resulting formulation was tested using various methods. In addition, the effect of components i.e. Vitamin E and Pluronic F-68 on the formulation was also studied.     

Results of satellite tagging of Atlantic bluefin tuna, <Emphasis Type="Italic">Thunnus thynnus</Emphasis>, off the coast of Ireland

Pop-up satellite archival tags were attached to six Atlantic bluefin tuna (Thunnus thynnus) off the west coast of Ireland in autumn 2003 and 2004. The satellite tags measured pressure, ambient temperature and light for the term of deployment. Radio pop-up satellite endpoint positions, light and sea surface temperature estimations of geolocation indicate that two fish tagged minutes apart off the coast of County Donegal, migrated to the eastern and western Atlantic Ocean over the following 8 months. The two fish were 5218 km apart at the termination of the experiment. After tagging in September and popping up the following March and April, one fish had traveled to the western Atlantic while the other was located in the waters off the southwest coast of Portugal. A third fish tagged off the coast of County Donegal in October 2004 moved into the Mediterranean Sea and was caught by a fishing vessel southeast of Malta on 11 June 2005. The results link bluefin tuna feeding on European foraging grounds with known eastern breeding regions and western Atlantic waters.     

A genotype calling algorithm for the Illumina BeadArray platform

MOTIVATION: Large-scale genotyping relies on the use of unsupervised automated calling algorithms to assign genotypes to hybridization data. A number of such calling algorithms have been recently established for the Affymetrix GeneChip genotyping technology. Here, we present a fast and accurate genotype calling algorithm for the Illumina BeadArray genotyping platforms. As the technology moves towards assaying millions of genetic polymorphisms simultaneously, there is a need for an integrated and easy-to-use software for calling genotypes. RESULTS: We have introduced a model-based genotype calling algorithm which does not rely on having prior training data or require computationally intensive procedures. The algorithm can assign genotypes to hybridization data from thousands of individuals simultaneously and pools information across multiple individuals to improve the calling. The method can accommodate variations in hybridization intensities which result in dramatic shifts of the position of the genotype clouds by identifying the optimal coordinates to initialize the algorithm. By incorporating the process of perturbation analysis, we can obtain a quality metric measuring the stability of the assigned genotype calls. We show that this quality metric can be used to identify SNPs with low call rates and accuracy. AVAILABILITY: The C++ executable for the algorithm described here is available by request from the authors.     

Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus)

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.     

C1q Protein Binds to the Apoptotic Nucleolus and Causes C1 Protease Degradation of Nucleolar Proteins

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r₂C1s₂), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus.     

Structural studies of biologically active glycosylated polyamidoamine (PAMAM) dendrimers

The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimer’s surface for interaction with the biological target.     

First somatic mutation of E2F1 in a critical DNA binding residue discovered in well-differentiated papillary mesothelioma of the peritoneum

Background

Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP.

Results

WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector.

Conclusions

The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.