Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.     

PRODUCTION OF 7-DEOXY-OKADAIC ACID BY A NEW CALEDONIAN STRAIN OF PROROCENTRUM LIMA (DINOPHYCEAE)

7-Deoxy-okadaic acid and okadaic acid were identified as the major diarrhetic shellfish poisoning (DSP) toxins produced by a New Caledonian strain of Prorocentrum lima Ehrenberg. Dinophysistoxin-1 was not produced by this strain. The cellular concentrations of 7-deoxy-okadaic acid were about one tenth that of okadaic acid and were maximal (∼1.4 pg·cell ^{− 1}) during the stationary growth phase of batch culture. Autolytic hydrolysis of cell extracts did not increase the concentrations of 7-deoxy-okadaic acid, whereas okadaic acid production increased more than 4-fold, indicating that 7-deoxy-okadaic acid, unlike okadaic acid, is not directly derived from large sulfated precursors. 7-Deoxy-okadaic acid could be detected by liquid chromatography-selected reaction monitoring mass spectrometry, HPLC-fluorescence detection after derivatization with 9-anthryldiazomethane (ADAM), and inhibition of protein phosphatases. The solvent washes currently used for solid-phase clean-up of ADAM-derivatized DSP samples elute derivatized 7-deoxy-okadaic acid, indicating that the current sample clean-up protocol for HPLC-fluorescence detection would miss any contamination by this toxin.     

First cervical vertebra (atlas) fracture mechanism studies using finite element method.

Injury mechanisms and stress distribution patterns are important in the clinical evaluation of spinal injuries. Recognition and interpretation of the failure patterns help to determine spinal instability and consequently the choice of treatment. Although, the biomechanics responses of the atlas have received much attention, it has not been investigated using theoretical modeling. Mathematical techniques such as finite element model will provide further understanding to the injury mechanisms of the atlas, which is important for the prevention, diagnosis, and treatment of spinal injuries. In the present study, a detailed three-dimensional finite element model of the human atlas (C1) was constructed, with the geometrical data obtained using a three-dimensional digitizer. Anterior arch, superior/inferior articular processes, transverse processes, posterior arch and posterior tubercule were modeled using eight-noded brick elements. Using the material properties from literature, the 7808-finite element model was exercised under three simulated axial compressive mode of pressure loading and boundary conditions to investigate the sites of failure reported in vivo and in vitro. This report demonstrates high concentration of localized stress at the anterior and posterior archs of the atlas, which agrees well with those reported in the literature. Furthermore, under simulated hyperextension, our results agreed well with the experimental findings, which show that the groove of the posterior arch is subjected to enormous bending moment. The close agreement of the failure location provided confidence to perform further analysis and in vitro experiments. These results may be potentially used to supplement experimental research in understanding the clinical biomechanics of the atlas.     

Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such "membrane-inserted" form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKC(alpha), the C2-V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.     

Nutrient uptake relationship to root characteristics of rice

Data on root parameters and distribution are important for an improved understanding of the factors influencing nutrient uptake by a crop. Therefore, a study was conducted on a Crowley silt loam at the Rice Research and Extension Center near Stuttgart, Arkansas to measure root growth and N, P and K uptake by three rice (Oryza sativa L.) cultivars at active tillering (36 days after emergence (DAE)), maximum tillering (41 DAE), 1.25 cm internode elongation (55 DAE), booting (77 DAE) and heading (88 DAE). Soil-root core samples were taken to a depth of 40 cm after plant samples were removed, sectioned into 5 cm intervals, roots were washed from soil and root lengths, dry weights and radii were measured. Root parameters were significantly affected by the soil depth × growth stage interaction. In addition, only root radius was affected by cultivar. At the 0- to 5-cm soil depth, root length density ranged from 38 to 93 cm cm^-3 throughout the growing season and decreased with depth to about 2 cm cm^-3 in the 35- to 40-cm depth increment. The increase in root length measured with each succeeding growth stage in each soil horizon also resulted in increased root surface area, hence providing more exposed area for nutrient uptake. About 90% of the total root length was found in the 0- to 20-cm soil depth throughout the season. Average root radius measured in the 0- to 5-cm and 35- to 40-cm depth increments ranged from 0.012 to 0.013 cm and 0.004 to 0.005 cm, respectively throughout the season. Total nutrient uptake by rice differed among cultivars only during vegetative growth. Differences in total nutrient uptake among the cultivars in the field appear to be related to absorption kinetics of the cultivars measured in a growth chamber study. Published with permission of the Arkansas Agricultural Experiment Station.     

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.     

Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage- and Histone Methylation-Dependent Pathways

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Analyses of soil bacterial diversity of the Schirmacher Oasis,Antarctica

Schirmacher Oasis, Antarctica, is a region with relatively large exposed area and consisted of many freshwater lakes. Nevertheless, only a few studies were done on the bacterial diversity of this region. Hence, this project was undertaken to determine the bacterial community in soil samples collected from the Schirmacher Oasis using the denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. A total of 79 partial 16S rDNA sequences were obtained from the excised DGGE bands, which corresponded to 63 different operational taxonomic units (OTUs) representing bacteria from seven different phyla. The most dominant phyla in descending order were Acidobacteria, Proteobacteria, Bacteroidetes, and Actinobacteria, Planctomycetes, Cyanobacteria and BRC1. There were 5.4 % of unclassified bacteria which cannot be grouped into any of the existing phyla. Eighty-seven percent of the OTUs had highest similarity with the uncultured bacteria from the NCBI GenBank database. Thirty-two percent of the OTUs were similar to bacteria reported in other parts of the Antarctica, while the others were related to bacteria found elsewhere outside the Antarctic.     

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.     

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.