Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Intralymphatic administration of BCG in melanoma patients

Summary From November 1973 to December 1974, 20 patients with advanced malignant melanoma were treated with BCG given by intralymphatic route at the Cancer Institute of Milan. The lyophilized Pasteur BCG was used. Patients were treated with a single dose ranging from 0.2–80 mg. Patients' performance status was never severely impaired.The most frequent side effects were fever, lymphangitis, and lymph node enlargement.Variations were observed in white cell count, ERS and immunoglobulins; in no case did we find evidence of liver toxicity or tumor growth enhancement. It is concluded that the intralymphatic route is a safe way of administrating BCG.     

E-cadherin deregulation in breast cancer

E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.     

Potential inter-guild interactions to enhance biological control of Bactericera cockerelli on tomatoes: a laboratory and cage study

The tomato–potato psyllid (TPP) Bactericera cockerelli, is a serious pest of solanaceous crops. Some populations are becoming pesticide-resistant, increasing the need for alternatives such as biological control (BC). This approach may be improved by combining different species of BC agents. We conducted three separate experiments to test four BC agents, either alone or combined with others: (1) A laboratory assay to test the effect of buckwheat (Fagopyrum esculentum) and alyssum (Lobularia maritima) flowers on the longevity of females of the parasitic wasp Tamarixia triozae; (2) A no-choice laboratory assay to investigate the consumption of B. cockerelli life stages by the predatory bug Engytatus nicotianae; (3) A cage experiment in a greenhouse to assess four natural enemy species against B. cockerelli on tomatoes: these were the predators Cleobora mellyi, Amblydromalus limonicus, E. nicotianae, and T. triozae. Access to buckwheat flowers allowed female T. triozae to live for an average of 10.9 days compared to 2.1 days with alyssum and 1.4 day with water but did not improve the BC of B. cockerelli. Adult E. nicotianae preyed on all offered B. cockerelli stages. In experiment 3, combinations of T. triozae with A. limonicus or E. nicotianae were not significantly better than single natural enemy species, except for the reduction of nymphal populations when A. limonicus and T. triozae were combined. Although there were few significant reductions in numbers of TPP when using natural enemy species combinations, some species showed good potential when used alone. We suggest testing earlier release of combinations of natural enemy for evaluate its impact on TPP.

     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis

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Understanding Genetic Diversity and Population Structure of a Poa pratensis Worldwide Collection through Morphological,Nuclear and Chloroplast Diversity Analysis

Poa pratensis L. is a forage and turf grass species well adapted to a wide range of mesic to moist habitats. Due to its genome complexity little is known regarding evolution, genome composition and intraspecific phylogenetic relationships of this species. In the present study we investigated the morphological and genetic diversity of 33 P. pratensis accessions from 23 different countries using both nuclear and chloroplast molecular markers as well as flow cytometry of somatic tissues. This with the aim of shedding light on the genetic diversity and phylogenetic relationships of the collection that includes both cultivated and wild materials. Morphological characterization showed that the most relevant traits able to distinguish cultivated from wild forms were spring growth habit and leaf colour. The genome size analysis revealed high variability both within and between accessions in both wild and cultivated materials. The sequence analysis of the trnL-F chloroplast region revealed a low polymorphism level that could be the result of the complex mode of reproduction of this species. In addition, a strong reduction of chloroplast SSR variability was detected in cultivated materials, where only two alleles were conserved out of the four present in wild accessions. Contrarily, at nuclear level, high variability exist in the collection where the analysis of 11 SSR loci allowed the detection of a total of 91 different alleles. A Bayesian analysis performed on nuclear SSR data revealed that studied materials belong to two main clusters. While wild materials are equally represented in both clusters, the domesticated forms are mostly belonging to cluster P2 which is characterized by lower genetic diversity compared to the cluster P1. In the Neighbour Joining tree no clear distinction was found between accessions with the exception of those from China and Mongolia that were clearly separated from all the others.     

Reproducibility of Volumetric Computed Tomography of Stable Small Pulmonary Nodules with Implications on Estimated Growth Rate and Optimal Scan Interval

Purpose

To use clinically measured reproducibility of volumetric CT (vCT) of lung nodules to estimate error in nodule growth rate in order to determine optimal scan interval for patient follow-up.

Methods

We performed quantitative vCT on 89 stable non-calcified nodules and 49 calcified nodules measuring 3–13 mm diameter in 71 patients who underwent 3–9 repeat vCT studies for clinical evaluation of pulmonary nodules. Calculated volume standard deviation as a function of mean nodule volume was used to compute error in estimated growth rate. This error was then used to determine the optimal patient follow-up scan interval while fixing the false positive rate at 5%.

Results

Linear regression of nodule volume standard deviation versus the mean nodule volume for stable non-calcified nodules yielded a slope of 0.057±0.002 (r² = 0.79, p<0.001). For calcified stable nodules, the regression slope was 0.052±0.005 (r² = 0.65, p = 0.03). Using this with the error propagation formula, the optimal patient follow-up scan interval was calculated to be 81 days, independent of initial nodule volume.

Conclusions

Reproducibility of vCT is excellent, and the standard error is proportional to the mean calculated nodule volume for the range of nodules examined. This relationship constrains statistical certainty of vCT calculated doubling times and results in an optimal scan interval that is independent of the initial nodule volume.     

A comparative stereological study of the term placenta in the donkey, pony and Thoroughbred

The aim of the study was to compare horse and donkey placentae using stereological techniques. Term placentae were collected at spontaneous foaling from seven Thoroughbred mares, seven pony mares, and six jenny donkeys. Maternal and foal weights were recorded and the mass, volume, and gross area of each allantochorion was also recorded. Ten random biopsies were recovered and processed for light microscopy from which the surface density of the microcotyledons (S_v) and the total microscopic area of fetomaternal contact were calculated stereologically. Gestation length was longer in the donkeys than the other two groups (median values: 371 vs. 327 and 341 days, P < 0.05). There were significant correlations between foal birthweight and gross area (rho = 0.89; n = 20; P < 0.05), mass (rho = 0.84; n = 20; P < 0.05) and volume (rho = 0.89; n = 20; P < 0.05) of the allantochorion. S_v was higher in the donkey placenta than the other groups (median values: 0.05 vs. 0.03 and 0.04 μm⁻¹, P < 0.05) although placental efficiency was lower in the donkeys (median values: 0.87 vs. 1.33 and 1.32 kg/m², P < 0.01). The results of the study confirmed that, although strong morphological similarities exist between the allantochorion of the horse and donkey, that of the donkey develops more complex microcotyledons, as judged stereologically, and exhibits a lower placental efficiency. These differences may be related to maternal genotype and/or the longer gestation length shown by the donkey compared to the horse, but a negative correlation (rho = −0.92, P < 0.01) was also found between age and placental efficiency in donkeys.     

Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.