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.     

Action of histamine on the rapidly adapting airway receptors in the dog

The effects of histamine on the activity of rapidly adapting receptors (RAR) of the airways were investigated in anesthetized dogs. With bolus injections given into the right atrium, the threshold dose of histamine required for the excitation of RAR (n = 7) was 0.82 microgram/kg (+1.33/-0.51, geometric mean). With increasing doses of histamine, a dose-response relationship was seen in the activity of RAR. Obstruction of the lymphatic drainage from the lungs reduced the threshold dose to histamine (i.e., shifted the dose-response curve to the left significantly). This change in the dose-response relationship was not accompanied by a corresponding change in the relationship of histamine dose to airway pressures recorded before and after lymphatic obstruction. Against a background of pulmonary venous congestion produced by partial obstruction of the mitral valve, subthreshold doses of histamine stimulated the RAR (n = 4). The excitatory effect of histamine on RAR was found to be abolished by the administration of the H1 receptor antagonist diphenhydramine but not by the H2 receptor antagonist cimetidine. Intravenous infusion of histamine (0.4 microgram.kg-1.min-1) for a period of 10 min increased the RAR activity (n = 6) significantly without producing detectable changes in airway mechanics. The results indicate that contraction of the smooth muscle of the airways may not be a prerequisite for the excitation of RAR, especially at low doses. It is suggested that some of the effects of histamine on RAR are mediated by a local expansion of the extravascular fluid caused by an increase in the permeability of the bronchial vasculature.     

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.     

Species preservations in an optimal harvest model with random prices

In this paper, we consider an optimal harvest model in which the objective is to maximize the expected return. The unit price of biomass is assumed constant until a random time when the price increases by a given amount. Furthermore, due to obvious environmental protection requirements, it is assumed that the fishery population is bounded from below for all time so as to reduce the danger of species extinction. Clearly, this problem is an optimal control problem in which a random parameter is involved. However, due to its special structure, it is shown that the problem is convertible into a deterministic optimal control problem and hence is solvable by an existing optimal control software package, MISER. The practical implication of several computed results obtained by this approach is discussed. They are also compared with other related results in the literature.     

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.     

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.     

Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing.