Distinguishing Antimicrobial Models with Different Resistance Mechanisms via Population Pharmacodynamic Modeling

Semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) modeling is increasingly used for antimicrobial drug development and optimization of dosage regimens, but systematic simulation-estimation studies to distinguish between competing PD models are lacking. This study compared the ability of static and dynamic in vitro infection models to distinguish between models with different resistance mechanisms and support accurate and precise parameter estimation. Monte Carlo simulations (MCS) were performed for models with one susceptible bacterial population without (M1) or with a resting stage (M2), a one population model with adaptive resistance (M5), models with pre-existing susceptible and resistant populations without (M3) or with (M4) inter-conversion, and a model with two pre-existing populations with adaptive resistance (M6). For each model, 200 datasets of the total bacterial population were simulated over 24h using static antibiotic concentrations (256-fold concentration range) or over 48h under dynamic conditions (dosing every 12h; elimination half-life: 1h). Twelve-hundred random datasets (each containing 20 curves for static or four curves for dynamic conditions) were generated by bootstrapping. Each dataset was estimated by all six models via population PD modeling to compare bias and precision. For M1 and M3, most parameter estimates were unbiased (<10%) and had good imprecision (<30%). However, parameters for adaptive resistance and inter-conversion for M2, M4, M5 and M6 had poor bias and large imprecision under static and dynamic conditions. For datasets that only contained viable counts of the total population, common statistical criteria and diagnostic plots did not support sound identification of the true resistance mechanism. Therefore, it seems advisable to quantify resistant bacteria and characterize their MICs and resistance mechanisms to support extended simulations and translate from in vitro experiments to animal infection models and ultimately patients.     

Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85.

Insulin-like growth factor-I (IGF-I) stimulates the production of 3-inositides and markedly increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by anti-phosphotyrosine antibodies, a portion of which is also associated with the IGF-I receptor. In this study, recombinant p85, the regulatory subunit of phosphatidylinositol 3-kinase, and fusion proteins containing various subdomains were used to investigate the association of p85 with the IGF-I receptor and to demonstrate that p85 is a direct in vitro substrate of the IGF-I receptor kinase. Solubilized IGF-I receptor was immobilized on antireceptor antibody-agarose beads. Following in vitro receptor phosphorylation and incubation with cell lysate, immobilized receptor became associated with phosphatidylinositol 3-kinase activity and with protein bands with molecular masses of 85 and 110 kDa, which correspond to the known molecular masses of the subunits of phosphatidylinositol 3-kinase. These associations were inhibited by the addition of recombinant intact p85 or SH2-containing fusion proteins, but not by fusion proteins containing its SH3 domain or breakpoint cluster homology region. A fusion protein containing the SH2 domains of Ras GTPase-activating protein also inhibited the association of phosphatidylinositol 3-kinase activity with immobilized IGF-I receptor, although less effectively than p85, whereas a similar construct containing the SH2 domain of pp60src was without effect. When immobilized phosphorylated IGF-I receptor was incubated with intact p85 or the SH2-containing fusion proteins, it became associated with and phosphorylated these proteins. These results demonstrate that at least in vitro, a tight association occurs between phosphorylated IGF-I receptor and phosphatidylinositol 3-kinase, that the region of phosphatidylinositol 3-kinase that contains its SH2 domains is directly involved in this association, and that this region is a direct substrate for IGF-I receptor tyrosine kinase. Furthermore, these results suggest that Ras GTPase-activating protein can also interact with the IGF-I receptor and that different SH2 domain-containing proteins interact with the IGF-I receptor with widely differing affinities.     

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.     

Excavating ghost footprints and tangled trees from modern genomes

Due to pervasive gene flow and admixture, simple bifurcating trees often do not provide an accurate representation of relationships among diverging lineages, but limited resolution in the available genomic data and the spatial distribution of samples has hindered detailed insights regarding the evolutionary and demographic history of many species and populations. In this issue of Molecular Ecology, Foote et al. (2019) combine a powerful sampling design with novel analytical methods adopted from human genetics to describe previously unrecognized patterns of recurrent vicariance and admixture among lineages in the globally distributed killer whale (Orcinus orca). Based on sequence data from modern samples alone, they discover clear signatures of ancient admixture with a now extinct “ghost” lineage, providing one of the first accounts of archaic introgression in a nonhominid species. Coupling a cost‐effective sequencing strategy with novel analytical approaches, their paper provides a roadmap for advancing inference of evolutionary history in other nonmodel species, promising exciting times ahead for our field.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Visiting bags: a labile thermal environment.

OBJECTIVE--To define usual colour and site of storage of visiting bags in general practitioners'' cars and to investigate effect of these variables on temperature inside bag. DESIGN--Questionnaire to general practitioners; serial temperature measurements from paired black visiting bags at different storage sites and from bags of different colour. SETTING--South Devon coastal town during May and June. SUBJECTS--200 general practitioners, of whom 145 returned legible questionnaires. MAIN OUTCOME MEASURES--Bag colour, duration and site of storage, temperature inside black bags at defined storage sites, and effects of bag colour on internal temperature. RESULTS--111 (77%) of the general practitioners carried a black visiting bag, and 76 kept their bag in their car all day. The bag was coolest in the car boot, but irrespective of storage site, maximum internal temperature of the bag was always over 25 degrees C and reached up to 80 degrees C. Spraying a black bag silver significantly reduced the bag''s internal temperature (mean difference 8.37 degrees C (95% confidence interval 6.68 to 9.86 degrees C) df = 59, t = 10.29, P < 0.001). CONCLUSIONS--General practitioners should use a silver coloured visiting bag; when visiting, they should store it in their car boot; at other times they should remove it to a cooler site.     

Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum

The soil fungus Trichoderma harzianum is a mycoparasitic fungus known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and fungal cell wall-degrading enzymes. These enzymes are believed to play an important role in mycoparasitism. Among the hydrolytic enzymes, we have identified a basic proteinase (Prb1) which is induced by either autoclaved mycelia, fungal cell wall preparation or chitin; however, the induction does not occur in the presence of glucose. The proteinase was purified and biochemically characterized as a serine proteinase of 31 kDa and pl 9.2. Based on the sequence of three internal peptides, synthetic oligonudeotide probes were designed. These probes allowed subsequent isolation of a cDNA and its corresponding genomic clone. The deduced amino acid sequence indicates that the proteinase is synthesized as a pre-proenzyme and allows its classification as a serine proteinase. Northen analysis shows that the induction of this enzyme is due to an increase in the corresponding mRNA level.     

NUMB does not impair growth and differentiation status of experimental gliomas

The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.     

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.