Power Estimation for Two-Sample Tests Using Importance and Antithetic Resampling

Collings and Hamilton (1988), described a uniform bootstrap method that is applied on observed or pilot data in order to approximate the power of the two-sample Wilcoxon test for location shift alternatives. In this paper we demonstrate how importance and antithetic resampling can be used to substantially reduce the amount of computation needed to approximate the power of the two-sample tests for location shift and scale alternatives. Importance and antithetic bootstrap resampling methods are applied to simulated data of different sample sizes from a variety of distributions as well as to data from the Iowa 65+ Rural Health Study. Also, a suggestion is given for using a combination of importance and antithetic resampling for approximating the power of two-sample tests.     

Targeting allosteric sites of human aromatase: a comprehensive in-silico and in-vitro workflow to find potential plant-based anti-breast cancer therapeutics

Recent findings suggested several allosteric pockets on human aromatase that could be utilised for the development of new modulators able to inhibit this enzyme in a new mechanism. Herein, we applied an integrated in-silico-based approach supported by in-vitro enzyme-based and cell-based validation assays to select the best leads able to target these allosteric binding sites from a small library of plant-derived natural products. Chrysin, apigenin, and resveratrol were found to be the best inhibitors targeting the enzyme’s substrate access channel and were able to produce a competitive inhibition with IC₅₀ values ranged from 1.7 to 15.8 µM. Moreover, they showed a more potent antiproliferative effect against ER+ (MCF-7) than ER- one (MDA-MB-231) cell lines. On the other hand, both pomiferin and berberine were the best hits for the enzyme’s haem-proximal cavity producing a non-competitive inhibition (IC₅₀ 15.1 and 21.4 µM, respectively) and showed selective antiproliferative activity towards MCF-7 cell lines.     

Effect of protein kinase C activation on agonist-mediated phosphositide metabolism in rabbit retinal cells

4β-Phorbol 12-myrisate 13-acetate (PMA), a tumour-promoting phorbol ester, and 1-oleoly-2-acetylglycerol (OAG), a synthetic diacylglycerol, induced an inhibition of muscarinic and ₁-adrenergic receptor-mediated stimulation of PIP₂ breakdown and IPs accumulation in both rabbit retinal slices and primary retinal cultures. Furthermore, an increase in [Ca²⁺]_i, mediated by activation of these receptors in 3–5 and 25–30 day old rabbit retinal cultures, was also inhibited by PMA. Neither PMA nor OAG had an effect on the serotonin-mediated PIP₂ breakdown, IPs accumulation or Ca²⁺ mobilization. Although A23187 also stimulated IPs formation by acting directly on phospholipase C, PMA had no effect. Maximal inhibition of the carbachol- and noradrenaline-mediated responses was achieved with a 15 min preincubation with PMA at concentrations of 0.1 and 0.01 μM in retinal slices and primary retinal cultures, respectively. Neither PMA nor OAG influenced the basal levels of phosphoinositides, IPs or [Ca²]_i. In addition, the inactive phorbol ester, 4-phorbol 12,13-didecanoate, had no effect on any of the agonist-induced responses. Staurosporine, a potent inhibitor of protein kinase C, significantly attenuated the inhibitory effects exerted by PMA and OAG. These results suggest that calcium- and phospholipid-dependent protein kinase, which is activated by either PMA or OAG, exert inhibitory effects on muscarinic and ₁-adrenergic responses. This modulatory feedback “down regulation” role by PKC does not, however, affect serotonergic mediated responses, and thus exhibits a certain selectivity about the site of action. The possible mechanism(s) by which PKC induces its actions are discussed.     

Structural and functional characterization of complement C4 and C1s-like molecules in teleost fish: insights into the evolution of classical and alternative pathways

There is growing evidence that certain components of complement systems in lower vertebrates are promiscuous in their modes of activation through the classical or alternative pathways. To better understand the evolution of the classical pathway, we have evaluated the degree of functional diversification of key components of the classical and alternative pathways in rainbow trout, an evolutionarily relevant teleost species. Trout C4 was purified in two distinct forms (C4-1 and C4-2), both exhibiting the presence of a thioester bond at the cDNA and protein levels. C4-1 and C4-2 bound in a similar manner to trout IgM-sensitized sheep erythrocytes in the presence of Ca(2+)/Mg(2+), and both C4 molecules equally restored the classical pathway-mediated hemolytic activity of serum depleted of C3 and C4. Reconstitution of activity was dependent on the presence of both C3-1 and C4-1/C4-2 and on the presence of IgM bound to the sheep erythrocytes. A C1s-like molecule was shown to cleave specifically purified C4-1 and C4-2 into C4b, while failing to cleave trout C3 molecules. The C1s preparation was unable to cleave trout factor B/C2 when added in the presence of C3b or C4b molecules. Our results show a striking conservation of the mode of activation of the classical pathway. We also show that functional interchange between components of the classical and alternative pathway in teleosts is more restricted than was anticipated. These data suggest that functional diversification between the two pathways must have occurred shortly after the gene duplication that gave rise to the earliest classical pathway molecules.     

Antagonist Microorganisms with the Ability to Control <Emphasis Type="Italic">Pythium</Emphasis> Damping-off of Tomato Seeds in Rockwool

A total of 237 microorganisms were isolated from five different greenhouse tomato growing media. Of those, 40 microorganisms reduced the in vitro mycelial growth of both Pythium aphanidermatum and Pythium ultimum. The ability of these microorganisms to control damping-off was then tested in rockwool. As a result, Pseudomonas corrugata strains 1 and 3, Pseudomonas fluorescens subgroup F and G strains 1, 2, 3, 4 and 5, Pseudomonas marginalis, Pseudomonas putida subgroup B strain 1, Pseudomonas syringae strain 1 and Pseudomonas viridiflava significantly reduced damping-off caused by P. ultimum or P. aphanidermatum. Pseudomonas marginalis was the only microorganism that significantly reduced damping-off caused by both pathogens.     

Poor competitive fitness of transgenically mitigated tobacco in competition with the wild type in a replacement series

Transgenic crops can interbreed with other crop cultivars or with related weeds, increasing the potential of the hybrid progeny for competition. To prevent generating competitive hybrids, we previously tested tobacco (Nicotiana tabacum L.) as a model for validating the transgenic mitigation (TM) concept using tandem constructs where a gene of choice is linked to mitigating genes that are positive or neutral to the crop, but deleterious to a recipient under competition. Here, we examine the efficacy of the TM concept at various ratios of transgenically mitigated tobacco in competition with the wild type tobacco in an ecological replacement series. The dwarf/herbicide-resistant TM transgenic plants cultivated alone under self-competition grew well and formed many more flowers than the tall wild type, which is an indication of greater reproductivity. In contrast to the wild type, TM flowering was almost completely suppressed in mixed cultures at most TM/wild type ratios up to 75% transgenic, as the TM plants were extremely unfit to reproduce. In addition, homozygous TM progeny had an even lower competitive fitness against the wild type than hemizygous/homozygous TM segregants. Thus, the TM technology was effective in reducing the risk of transgene establishment of intraspecific transgenic hybrids at different competitive levels, at the close spacing typical of weed populations.     

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.     

Evaluating postoperative analgesics in mice using telemetry

The study examined the efficacy of preemptive or postoperative analgesia on surgical pain in the mouse. Radiotelemetry transmitters were surgically implanted in 28 female ICR mice. A mock ova implantation surgery was then performed. Mice were treated with a single dose of buprenorphine or flunixin meglumine prior to or after surgery, three doses of buprenorphine, or were untreated. Heart rate, blood pressure, home cage activity, food and water consumption, and body weight were measured. The no-analgesia group showed no significant differences between any parameters collected prior to surgery and those collected at similar times during the day of surgery. Significant increases in mouse activity on the day of surgery occurred with all analgesic treatments, compared with pre-surgical activity. There were no consistent significant changes in any other telemetry parameter after treatment with analgesics compared with no analgesia. Food consumption and body weight the day after surgery were reduced significantly in the animals treated with three doses of buprenorphine compared with untreated mice and mice given a single dose of buprenorphine. We conclude that the mock ova implant procedure does not induce sufficient pain to cause alterations in heart rate and blood pressure in the mouse. Activity was significantly reduced in the first 6 h after surgery in mice without analgesia, compared with activity prior to surgery. There were no significant differences between pre-emptive and postoperative analgesia. Body weight and food and water consumption were poor measures of pain because analgesia alone affected these parameters.     

Broad-host-range mobilizable suicide vectors for promoter trapping in gram-negative bacteria

Here we report the construction of three different vectors for the identification of bacterial genes induced in vitro and/or in vivo. These plasmids contain kanamycin, gentamicin, or tetracycline resistance genes as selectable markers. A promoterless cat and an improved GFP (mut3-gfp) can be used to follow the induction of gene expression by measuring chloramphenicol resistance and fluorescence, respectively.     

Comparative Analysis Between Pseudomonas aeruginosa Genotypes and Severity of Symptoms in Patients with Unilateral or Bilateral Otitis Externa

Random amplified polymorphic DNA (RAPD) analysis was done on 32 isolates of Pseudomonas aeruginosa. These isolates were obtained from 22 patients who presented to the emergency room in a major medical center in Beirut, Lebanon, during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients had yellowish to greenish discharge, moderate to severe external auditory canal swelling, moderate to severe pain, and periauricular cellulitis. None of these patients had intrinsic predisposing factors. An ear swab was obtained from both ears of patients, cultured on trypticase soy agar. P. aeruginosa was identified on the basis of pyocyanine production and API identification kits. RAPD analysis was done by using two primers (10 mer and 21 mer primers) and appropriate PCR conditions on extracted DNA. Our data have shown 23 RAPD patterns (A–W) distributed among the 32 P. aeruginosa isolates. RAPD patterns were reproducible. Twenty of 32 isolates were recovered from 10 patients with bilateral otitis externa. The remaining 12 of 32 isolates were recovered from 12 different patients with unilateral otitis externa. Eleven RAPD patterns (A,B,C,D,E,F,H,I,R,U,V) were associated with severe clinical symptoms, including severe pain, severe external auditory canal swelling, periauricular cellulitis, and a yellowish discharge. The remaining RAPD patterns were not associated with severe infections. This denotes a possible association between certain genotypes and severity of symptoms. Received: 6 July 2000 / Accepted: 5 September 2000